Methods for evaluating, monitoring, and modulating aging process

ABSTRACT

Disclosed herein are methods of increasing the expression rate of epigenetic markers such as ELOVL2, KLF14, and PENK with administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof). Also described herein are methods of modulating the methylation pattern of epigenetic markers such as ELOVL2, KLF14, and PENK with administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof).

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No. 15/610,407, filed May 31, 2017, which claims the benefit of U.S. Provisional Application No. 62/343,752, filed on May 31, 2016, each of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 26, 2017, is named 49697-706_301_SL.txt and is 28,997 bytes in size.

BACKGROUND OF THE DISCLOSURE

The rate and progression of aging varies from person to person and are further influenced by environmental factors, lifestyle choices, and/or physical fitness. In some instances, studies have shown that the state of the epigenome (e.g., mutation within the genome and/or methylation) correlate with age. As such, DNA methylation are utilized, for example, for determining age or changes in the rate of aging based on environmental factors, lifestyle choices, and/or physical fitness.

SUMMARY OF THE DISCLOSURE

Provided herein are therapeutic agents capable of increasing the gene expression of an epigenetic marker described herein. Also provided herein are therapeutic agents capable of decreasing the methylation level/status of an epigenetic marker described herein.

In some embodiments, disclosed herein is a method of increasing the expression rate of genes: ELOVL2, KLF14, PENK, or a combination thereof in a first subject, comprising: (a) administering to the first subject a therapeutically effective dose of a therapeutic agent for a first time period; (b) obtaining a sample from the first subject; and (c) determining whether the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased in the first subject relative to a control by contacting the sample with a probe that recognizes ELOVL2, KLF14, or PENK and detecting binding between ELOVL2, KLF14, or PENK and the probe.

In some embodiments, the therapeutic agent comprises vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof. In some embodiments, the therapeutic agent comprises vitamin C or its derivatives or pharmaceutically acceptable salts thereof. In some embodiments, the therapeutic agent is vitamin C. In some embodiments, the therapeutic agent is L-ascorbic acid 2-phosphate.

In some embodiments, the expression level of ELOVL2 gene is determined by contacting the sample with a probe that recognizes ELOVL2 and detecting binding between the probe and ELOVL2. In some embodiments, the expression level of KLF14 gene is determined by contacting the sample with a probe that recognizes KLF14 and detecting binding between the probe and KLF14. In some embodiments, the expression levels of ELOVL2 and KLF14 are determined by contacting the sample with a probe that recognizes ELOVL2 and a probe that recognizes KLF14 and detecting each respective binding between the probes and ELOVL2 and KLF14. In some embodiments, the expression levels of ELOVL2, KLF14, and PENK are determined.

In some embodiments, an increase in the expression rate of genes: ELOVL2, KLF14, PENK, or a combination thereof further correlates to a decrease in cell senescence.

In some embodiments, an increase in the expression rate of genes: ELOVL2, KLF14, PENK, or a combination thereof further correlates to an increase in cell proliferation.

In some embodiments, an increase in the expression rate of genes: ELOVL2, KLF14, PENK, or a combination thereof further correlates to an increase in cell survival.

In some embodiments, an increase in the expression rate of genes: ELOVL2, KLF14, PENK, or a combination thereof further correlates to a decrease in DNA methylation.

In some embodiments, an increase in the expression rate of genes: ELOVL2, KLF14, PENK, or a combination thereof leads to a methylation pattern that mimics the methylation pattern of a sample obtained from a second subject. In some embodiments, the second subject is younger in chronological age relative to the first subject. In some embodiments, the second subject is younger in chronological age relative to the first subject by at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 50 years, or more.

In some embodiments, the control comprises the expression level of genes: ELOVL2, KLF14, PENK, or a combination thereof obtained from a sample from the subject prior to administration of the therapeutic agent. In some embodiments, the control comprises a normalized expression level of ELOVL2, KLF14, PENK, or a combination thereof obtained from a set of samples without exposure to the therapeutic agent. In some embodiments, the set of samples are a set of cell samples.

In some embodiments, the method further comprises increasing the dose of the therapeutic agent if the expression level of genes: ELOVL2, KLF14, PENK, or a combination thereof has not increased relative to the control. In some embodiments, the method further comprises increasing the dose of the therapeutic agent if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased relative to the control and at a rate that is below a target range.

In some embodiments, the method further comprises decreasing or maintaining the dose of the therapeutic agent if the expression level of genes: ELOVL2, KLF14, PENK, or a combination thereof has increased relative to the control. In some embodiments, the method further comprises maintaining the dose of the therapeutic agent if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased relative to the control and at a rate that is within a target range. In some embodiments, the method further comprises decreasing the dose of the therapeutic agent if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased relative to the control and at a rate that is above a target range.

In some embodiments, the dose of the therapeutic agent is increased, decreased, or maintained for a second period of time prior to redetermining the expression level of genes: ELOVL2, KLF14, PENK, or a combination thereof.

In some embodiments, the first period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

In some embodiments, the second period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

In some embodiments, the method further comprises determining the expression level of FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, BDNF, NDF, GDNF, cortisol, or a combination thereof. In some embodiments, the method further comprises determining the expression level of FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or a combination thereof. In some embodiments, the method further comprises determining the expression level of an epigenetic marker selected from Table 1.

In some embodiments, provided herein is a method of modulating the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof in a first subject, comprising: (a) administering to the first subject a therapeutically effective dose of a therapeutic agent for a first time period; (b) obtaining a sample from the first subject; and (c) determining whether the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed in the first subject relative to a control by contacting the sample with a set of probes and detecting a set of hybridization products to determine the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof.

In some embodiments, the therapeutic agent comprises vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof. In some embodiments, the therapeutic agent comprises vitamin C or its derivatives or pharmaceutically acceptable salts thereof. In some embodiments, the therapeutic agent is vitamin C. In some embodiments, the therapeutic agent is L-ascorbic acid 2-phosphate.

In some embodiments, the sample is further treated with a deaminating agent prior to determining the methylation pattern.

In some embodiments, the methylation pattern of ELOVL2 is determined. In some embodiments, the methylation pattern of KLF14 is determined. In some embodiments, the methylation pattern of PENK is determined. In some embodiments, the methylation patterns of ELOVL2 and KLF14 are determined. In some embodiments, the methylation patterns of ELOVL2, KLF14, and PENK are determined.

In some embodiments, a change in the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof is a decrease in methylation status of ELOVL2, KLF14, PENK, or a combination thereof.

In some embodiments, a decrease in the methylation status of ELOVL2, KLF14, PENK, or a combination thereof further correlates to a decrease in cell senescence.

In some embodiments, a decrease in the methylation status of ELOVL2, KLF14, PENK, or a combination thereof further correlates to an increase in cell proliferation.

In some embodiments, a decrease in the methylation status of ELOVL2, KLF14, PENK, or a combination thereof further correlates to an increase in cell survival.

In some embodiments, a decrease in the methylation status of ELOVL2, KLF14, PENK, or a combination thereof leads to a methylation pattern that mimics the methylation pattern of a sample obtained from a second subject. In some embodiments, the second subject is younger in chronological age relative to the first subject. In some embodiments, the second subject is younger in chronological age relative to the first subject by at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 50 years, or more.

In some embodiments, the control comprises the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof obtained from a sample from the subject prior to administration of the therapeutic agent.

In some embodiments, the control comprises a normalized methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof obtained from a set of samples without exposure to the therapeutic agent. In some embodiments, the set of samples are a set of cell samples.

In some embodiments, the method further comprises increasing the dose of the therapeutic agent if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has not changed relative to the control. In some embodiments, the method further comprises increasing the dose of the therapeutic agent if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control and to a degree lower than a target range.

In some embodiments, the method further comprises decreasing or maintaining the dose of the therapeutic agent if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control. In some embodiments, the method further comprises maintaining the dose of the therapeutic agent if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control and to a degree within a target range. In some embodiments, the method further comprises decreasing the dose of the therapeutic agent if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control and to a degree above a target range.

In some embodiments, the dose of the therapeutic agent is increased, decreased, or maintained for a second period of time prior to redetermining the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof.

In some embodiments, the first period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

In some embodiments, the second period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

In some embodiments, the method further comprises determining the expression level of FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, BDNF, NDF, GDNF, cortisol, or a combination thereof. In some embodiments, the method further comprises determining the methylation pattern of FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or a combination thereof. In some embodiments, the method further comprises determining the methylation pattern of an epigenetic marker selected from Table 1.

In some embodiments, the therapeutically effective dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof comprises from about 0.1 μg/mL to about 200 μg/mL. In some embodiments, the therapeutically effective dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof comprises from about 1 μg/mL to about 150 μg/mL. In some embodiments, the therapeutically effective dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof comprises from about 5 μg/mL to about 100 μg/mL. In some embodiments, the therapeutically effective dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof comprises from about 10 μg/mL to about 100 μg/mL. In some embodiments, the therapeutically effective dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof comprises from about 20 μg/mL to about 100 μg/mL. In some embodiments, the therapeutically effective dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof comprises from about 30 μg/mL to about 100 μg/mL. In some embodiments, the therapeutically effective dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof comprises from about 50 μg/mL to about 100 μg/mL. In some embodiments, the therapeutically effective dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof comprises from about 1 μg/mL to about 50 μg/mL. In some embodiments, the therapeutically effective dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof comprises from about 5 μg/mL to about 50 μg/mL. In some embodiments, the therapeutically effective dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof comprises from about 10 μg/mL to about 50 μg/mL. In some embodiments, the therapeutically effective dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof comprises from about 50 μg/mL to about 200 μg/mL.

In some embodiments, a dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof greater than 200 μg/mL increases reactive oxidative species. In some embodiments, a dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof greater than 200 μg/mL leads to a methylation pattern that mimics the methylation pattern of a sample obtained from a third subject who is older in chronological age relative to the first subject.

In some embodiments, the third subject is older in chronological age relative to the first subject by at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 50 years, or more.

In some embodiments, the method further comprises administering to the first subject an additional therapeutic agent.

In some embodiments, the sample is a cell sample. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a tissue sample.

In some embodiments, the sample is obtained from a subject having a metabolic disease or condition. In some embodiments, the metabolic disease or condition comprises diabetes or pre-diabetes. In some embodiments, diabetes is type I diabetes. In some embodiments, diabetes is type II diabetes. In some embodiments, diabetes is type IV diabetes.

In some embodiments, the sample is obtained from a subject having a ELOVL2-associated disease or indication. In some embodiments, the sample is obtained from a subject having a KLF14-associated disease or indication. In some embodiments, the sample is obtained from a subject having a PENK-associated disease or indication.

In some embodiments, the sample is obtained from a subject having Werner syndrome.

In some embodiments, the sample is obtained from a subject having progeria.

In some embodiments, the sample is obtained from a subject having post-traumatic stress disorder.

In some embodiments, the sample is obtained from a subject having an elevated body mass index (BMI). In some embodiments, the elevated BMI is a BMI of 25 kg/m², 26 kg/m², 27 kg/m², 28 kg/m², 29 kg/m², 30 kg/m², 35 kg/m², 40 kg/m² or more.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the disclosure are set forth with particularity in the appended claims. The patent application file contains at least one drawing executed in color. Copies of this patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

FIG. 1A-FIG. 1H illustrate phenotypic and genotypic effects of concentration dependent vitamin C treatment were analyzed on WI38 PD46 and 48 fibroblast cells. FIG. 1A shows cell images of 12-well plate treated with low concentration vitamin C at Day 0, 4 and 5 for PD46. FIG. 1B shows confluency plot calculated through ImageJ of PD46, n=2. FIG. 1C and FIG. 1D illustrate expression graphs for ARM and SLC2A1 for PD46, n=3. FIG. 1E shows cell images of 12-well treated with high concentration vitamin C at Day 0, 4 and 5 for PD48. FIG. 1F shows confluency plot calculated through ImageJ of PD48, n=2. FIG. 1G and FIG. 1H show expression graphs for ARM and SLC2A1 for PD48, n=3.

FIG. 2A-FIG. 2J illustrate phenotypic and genotypic effects of vitamin C treatment were analyzed on younger WI38 PD42 and older WI38 PD58 fibroblasts. FIG. 2A shows cell images of 12-well at Day 0, 1 and 2 of treatment for PD42. FIG. 2B shows confluency plot calculated through ImageJ of PD42, n=2. FIG. 2C and FIG. 2D show expression graphs for ARM and SLC2A1 for PD42, n=3. FIG. 2E shows cell images of 12-well at Day 0, 5 and 7 of treatment for PD58. FIG. 2F shows confluency plot calculated through ImageJ of PD58, n=2. FIG. 2G and FIG. 2H illustrate expression graphs for ARM and SLC2A1 for PD53, n=3. FIG. 2I shows cell images of senescence and DAPI staining of PD45.5 fibroblasts. FIG. 2J shows graph of percentage senescence for younger PD32 fibroblast and older PD45.5 fibroblast. n=3.

FIG. 3A-FIG. 3D show phenotypic and genotypic effects of 6-O-Palmitoyl L-ascorbic acid treatment were analyzed on younger WI38 PD55 fibroblasts. FIG. 3A shows cell images of 12-well at Day 0 and Day 8 of treatment for PD55. FIG. 3B shows confluency plot calculated through ImageJ for PD55, n=2. FIG. 3C and FIG. 3D illustrate expression graph for ARM and SLC2A1 for PD55, n=3.

FIG. 4A-FIG. 4G show phenotypic and genotypic effects of dehydroascorbic acid and vitamin C treatment complemented with the addition of insulin were analyzed on WI38 PD54 fibroblast cells. FIG. 4A shows diagram of postulated pathway for interconversion of DHAA to vitamin C and their effect on fibroblast cells. FIG. 4B shows cell images of 12-well at Day 10 of treatment for PD54. FIG. 4C shows confluency plot calculated through ImageJ of PD54, n=2. FIG. 4D and FIG. 4E illustrate expression graphs for ARM and SLC2A1 for PD54 or PD55, respectively, n=3. FIG. 4F shows graph of percentage senescence of PD45 fibroblast. n=3. FIG. 4G shows fluorescent ROS assay showing fluorescent ROS relative to total fibroblasts in PD48 fibroblasts.

FIG. 5 illustrates that patients with diabetes have an older biological age than patients who do not have diabetes.

FIG. 6A-FIG. 6B show correlation of biological age with BMI and gender. FIG. 6A illustrates the correlation of BMI with biological age. FIG. 6B illustrates the correlation of biological aging between male and female.

FIG. 7 shows biological age prediction using an exemplary 71 methylation markers in three progeria cell lines. Each biological age (bioage) is higher than chronological age.

FIG. 8 shows that external influences, such as diet and exercise, reverse biological age in a 6 month trial.

FIG. 9 illustrates an exemplary list of genes and CpG sites that are utilized for biological age prediction.

FIG. 10 shows a decrease in expression of ELOVL2 and KLF14 in older fibroblasts.

FIG. 11A-FIG. 11B show decrease in expression of ELOVL2 in cell line IMR90 (FIG. 11A) and cell line WI38 (FIG. 11B).

FIG. 12A-FIG. 12C show the expression level of ELOVL2 and KLF14 in human blood (FIG. 12A), a human fibroblast cell line WI38 (FIG. 12B), and human lens tissue (FIG. 12C).

FIG. 13 shows the expression level of an exemplary list of genes.

FIG. 14A-FIG. 14C shows the biological age (or methylation age) increases with age. FIG. 14A shows the biological age increases with cell line population doubling. FIG. 14B shows the increase in methylation level of ELOVL2, PENK, and KLF14. FIG. 14C shows the increase in methylation level of FHL2 and SMC4.

FIG. 15 shows human KLF14 locus showing methylation CpG islands.

FIG. 16 shows human ELOVL2 locus showing methylation CpG islands.

FIG. 17A-FIG. 17C show ELOVL2 knockdown efficiency in three cell lines: WI38 (FIG. 17A), IMR90 (FIG. 17B), and 293T (FIG. 17C).

FIG. 18A-FIG. 18D show that ELOVL2 knockdown reduces cell proliferation. FIG. 18A shows a decrease of cells in ELOVL2 knockdown relative to the control (shLuc) in all three cell lines, WI38, IMR90, and 293T. FIG. 18B-FIG. 18D show the PD45 confluency of ELOVL2 knockdown relative to the control (shLuc) in the respective cell lines; WI38 (FIG. 18B), IMR90 (FIG. 18C), and 293T (FIG. 18D).

FIG. 19A-FIG. 19C show ELOVL2 knockdown increases senescence in cell lines: WI38 (FIG. 19A), IMR90 (FIG. 19B) and 293T (FIG. 19C).

FIG. 20 shows ELOVL2 overexpression increases survival in old cells (PD56).

FIG. 21 shows knockdown of KLF14 in WI38 cells.

FIG. 22 shows the effect of KLF14 knockdown on other genes. The KLF14 knockdown is about 99.5%.

FIG. 23 illustrates the morphology of knockdown of ELOVL2 and KLF14 in cells.

FIG. 24 shows a senescence assay of the knockdown cells.

FIG. 25A-FIG. 25C show WI38 PD55 confluency in the presence of different concentrations of vitamin C (FIG. 25A), L-dehydro ascorbic acid (DHAA or DHA) (FIG. 25B), or L-ascorbic acid 2-phosphate (VcP) (FIG. 25C).

FIG. 26 illustrates cell senescence of WI38 PD55 in the presence of different concentrations of vitamin C, L-dehydro ascorbic acid (DHAA or DHA), or L-ascorbic acid 2-phosphate (VcP).

FIG. 27 illustrates ELOVL2 expression in aging WI38 cells (PD55).

FIG. 28 shows reversal of biological age by reprogramming of aged fibroblast into iPSCs.

FIG. 29 shows the expressions of ELOVL2 in different mouse tissues.

FIG. 30 illustrates ELOVL2 expression in a mouse liver sample.

FIG. 31 illustrates ELOVL2 expression and senescence in a heterozygous knockout mouse model.

FIG. 32 illustrates a comparison of ELOVL2 and KLF14 methylation levels in the liver samples of young vs. aged mice.

FIG. 33A-FIG. 33B illustrate liver cell senescence in a 2-year old ELOVL2 heterozygous knockout mouse. FIG. 33A illustrates β-galactosidase staining of mouse liver cells Het 83-2, Het 77-1, and WT 81-5. Of the three types of cells tested, Het 83-2 exhibits the highest β-galactosidase activity (FIG. 33B).

FIG. 34 illustrates aging phenotypes associated with a Het 83-2 ELOVL2 heterozygous mouse. The mouse showed aging phenotypes such as hair loss, obesity, and tumor formation.

FIG. 35 shows the methylation age of 32 participants. Arrows going down (green): meditators with younger DNA at end of yoga intervention. Arrows going up (orange): meditators with older DNA at the end of yoga intervention. Blue line (dot) indicates meditator's calendar age.

FIG. 36 shows the salivary cortisol level at 30 minutes after meditation either taken prior to attendance of a yoga retreat (Anaadhi yoga retreat) or post attendance of the yoga retreat.

FIG. 37A-FIG. 37C show senescence and Elovl2 deletion affecting the spatial memory of mice in a Morris water maze. FIG. 37A shows the spatial memory performance of old wild type, young wild type, young Elovl2^(+/−), and young Elovl2^(−/−) mice. FIG. 37B and FIG. 37C show the frequency of platform crossing.

FIG. 38A-FIG. 38B show NAA/Cr and MI/Cr ratio, ADC, and Blood-perfusion (B-per) MRI analysis of wild type young (WT-Y) mice, wild type old (WT-O) mice, Elovl2 single (+/− Y) knock-out mice and Elovl2 double (−/− Y) knock-out mice. FIG. 38A shows the relative level in the hippocampus of the mice. FIG. 38B shows the relative level in the cortex of the mice.

DETAILED DESCRIPTION OF THE DISCLOSURE

Aging is a complex process that is characterized with a global decline in physiological functions and an increased risk for aging-related diseases or conditions. In some instances, the rate of aging correlates with the methylation status and/or expression levels of different epigenetic markers. As such in some cases, methylation status and/or expression levels of an epigenetic marker is utilized, for example, for determining or predicting the rate of aging of a subject; the progression, relapse, or refractory event of an aging-related disease or condition; or for monitoring the efficacy of a particular treatment option.

In some embodiments, disclosed herein is a method of retarding and/or reversing the biological age of a subject. In some instances, also described herein is a method of mimicking the biological age of a first subject to the biological age (e.g., an age based on the expression level or methylation profile of an epigenetic marker) of a second subject, in which the second subject is younger in chronological age (or actual age) than the first subject. In some cases, the method of retarding and/or reversing the biological age of a subject comprises administration to the subject a therapeutically effective dose of a therapeutic agent. In additional cases, the method of mimicking the biological age of a first subject to the biological age of a second subject comprises administration to the subject a therapeutically effective dose of a therapeutic agent.

In some instances, also described herein is a method of retarding and/or reversing the biological age of a subject suffering from a disease or condition. In some cases, the disease or condition is an aging-related disease or condition. In some cases, the method comprises administration to the subject suffering from a disease or condition a therapeutically effective dose of a therapeutic agent.

In some instances, additional described herein is a method of screening therapeutic agents to determine a therapeutic agent that is capable of retarding and/or reversing the biological age of a subject.

In some instances, also described herein include a method of reprogramming a cell to be transformed into an induced pluripotent stem cell (iPSC).

In additional instances, described herein include kits for use with one or more of the methods described herein.

Methods of Use

In some embodiments, disclosed herein is a method of retarding and/or reversing the biological age of a subject. In some instances, the method comprises increasing the expression rate or expression level of one or more epigenetic markers. In some instances, the one or more epigenetic markers are one or more genes. In some instances, the one or more epigenetic markers comprise ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or an epigenetic marker selected from Table 1. In some instances, the one or more epigenetic markers comprise ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or a combination thereof. In some cases, the one or more epigenetic markers comprise ELOVL2, KLF14, PENK, or a combination thereof.

In some embodiments, disclosed herein is a method of increasing the expression rate of ELOVL2, KLF14, PENK or a combination thereof in a first subject, comprising (a) administering to the first subject a therapeutically effective dose of a therapeutic agent for a first time period; (b) obtaining a sample from the first subject; and (c) determining whether the expression level of ELOVL2, KLF14, PENK or a combination thereof has increased in the first subject relative to a control by contacting the sample with a probe that recognizes ELOVL2, KLF14, or PENK and detecting binding between ELOVL2, KLF14, or PENK and the probe.

In some embodiments, the expression level of ELOVL2 is determined by contacting the sample with a probe that recognizes ELOVL2 and detecting binding between the probe and ELOVL2. In some cases, the expression level of KLF14 is determined by contacting the sample with a probe that recognizes KLF14 and detecting binding between the probe and KLF14. In some instances, the expression levels of ELOVL2 and KLF14 are determined by contacting the sample with a probe that recognizes ELOVL2 and a probe that recognizes KLF14 and detecting each respective binding between the probes and ELOVL2 and KLF14. In additional instances, the expression levels of ELOVL2, KLF14, and PENK are determined.

ELOVL fatty acid elongase 2 (ELOVL2) encodes a transmembrane protein involved in catalyzing the rate-limiting step of the long-chain fatty acids elongation cycle. In some instances, the methylation level or methylation status of ELOVL2 correlates to chronological age or the actual age of a subject (e.g., a human). For example, the methylation state or level of ELOVL2 increases as a subject ages. In some instances, biological age of a subject refers to the methylation level or methylation status of ELOVL2. In some cases, a CpG site within ELOVL2 comprises cg23606718, cg16867657, cg24724428, or cg21572722. In some cases, the biological age of a subject is based on the methylation level or status of cg23606718, cg16867657, cg24724428, and/or cg21572722. In some cases, the biological age of a subject is based on the methylation level or status of cg23606718 and/or cg16867657.

Furthermore, in some cases, the expression level of ELOVL2 decreases as a subject ages. In some cases, the biological age of a subject refers to the expression level of ELOVL2.

Kruppel-like factor 14 (KLF14), also known as basic transcription element-binding protein 5 (BTEB5), encodes a member of the Kruppel-like family of transcription factors. In some instances, KLF14 protein regulates the transcription of TGFβRII and is a master regulator of gene expression in adipose tissue. In some instances, the methylation level or methylation status of KLF14 correlates to chronological age or the actual age of a subject (e.g., a human). For example, the methylation state or level of KLF14 increases as a subject ages. In some instances, biological age of a subject refers to the methylation level or methylation status of KLF14. In some cases, a CpG site within KLF14 comprises cg14361627, cg08097417, cg07955995, cg20426994, cg04528819, cg09499629, and/or cg22285878. In some cases, the biological age of a subject is based on the methylation level or status of cg14361627, cg08097417, cg07955995, cg20426994, cg04528819, cg09499629, and/or cg22285878.

In some cases, the expression level of KLF14 decreases as a subject ages. In some cases, the biological age of a subject refers to the expression level of KLF14.

Proenkephalin (PENK) encodes a preproprotein that is proteolytically processed to generate multiple protein products. In some instances, the products of PENK comprise pentapeptide opioids Met-enkephalin and Leu-enkephalin. In some instances, the methylation level or methylation status of PENK correlates to chronological age or the actual age of a subject (e.g., a human). For example, the methylation state or level of PENK increases as a subject ages. In some instances, biological age of a subject refers to the methylation level or methylation status of PENK. In some cases, a CpG site within PENK comprises cg16419235. In some cases, the biological age of a subject is based on the methylation level or status of cg16419235.

In some cases, the expression level of PENK decreases as a subject ages. In some cases, the biological age of a subject refers to the expression level of PENK.

In some instances, the method further comprises determining the expression level of FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or a combination thereof.

In some cases, the method additionally comprises determining the expression level of an epigenetic marker selected from Table 1.

In some embodiments, a neurotrophin is correlated with the biological age of a subject. In some instances, the expression level of a neurotrophin is correlated with the biological age of a subject. In some cases, the expression level is an elevated expression level. In some instances, the neurotrophin is brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), or glial cell-derived neurotrophic factor (GDNF). BDNF is involved in supporting the survival of existing neurons and participate in the growth and differentiation of new neurons and synapses. NGF, similar to BDNF, is involved in the development and phenotypic maintenance of neurons in the peripheral nervous system (PNS) and the functional integrity of cholinergic neurons in the central nervous system (CNS). GDNF is involved in promoting the survival and differentiation of dopaminergic neurons.

In some instances, disclosed herein is a method of increasing the expression rate or level of a neurotrophin in a subject, comprising administering to the subject a therapeutically effective dose of a therapeutic agent for a first time period, obtaining a sample from the subject, and determining whether the expression level or rate of the neurotrophin has increased in the subject relative to a control by contacting the sample with a probe that recognizes the neurotrophin and detecting binding between the neurotrophin and the probe. In some cases, the neurotrophin is BDNF, NGF, or GDNF. In some cases, a method described herein comprises increasing the expression rate or level of BDNF gene in a subject, comprising administering to the subject a therapeutically effective dose of a therapeutic agent for a first time period, obtaining a sample from the subject, and determining whether the expression level or rate of BDNF gene has increased in the subject relative to a control by contacting the sample with a probe that recognizes BDNF and detecting binding between BDNF and the probe. In some cases, a method described herein comprises increasing the expression rate or level of NGF gene in a subject, comprising administering to the subject a therapeutically effective dose of a therapeutic agent for a first time period, obtaining a sample from the subject, and determining whether the expression level or rate of NGF gene has increased in the subject relative to a control by contacting the sample with a probe that recognizes NGF and detecting binding between NGF and the probe. In some cases, a method described herein comprises increasing the expression rate or level of GDNF gene in a subject, comprising administering to the subject a therapeutically effective dose of a therapeutic agent for a first time period, obtaining a sample from the subject, and determining whether the expression level or rate of GDNF gene has increased in the subject relative to a control by contacting the sample with a probe that recognizes GDNF and detecting binding between GDNF and the probe. In some cases, an elevated expression level of BDNF is correlated with a biological age that is younger than the chronological age (or actual age) of the subject. In some cases, an elevated expression level of NGF is correlated with a biological age that is younger than the chronological age (or actual age) of the subject. In some cases, an elevated expression level of GDNF is correlated with a biological age that is younger than the chronological age (or actual age) of the subject.

In some embodiments, a cortisol level is correlated with the biological age of a subject. Cortisol is a steroid hormone, under the glucocorticoid class of hormones. It is produced by the zona fasciculata of the adrenal cortex within the adrenal gland. In some instances, cortisol, which activates glucocorticoid receptors that act as transcription factors, modulate DNA methylation levels. In such cases, the DNA methylation is genome-wide DNA methylation.

In some instances, an elevated cortisol level is observed with administration of a therapeutically effective dose of a therapeutic agent to a subject. In some cases, the elevated cortisol level modulates the DNA methylation level, in which the methylation level subsequently correlates with a biological age of the subject that is younger than the chronological age (or actual age) of the subject.

In some instances, the therapeutic agent comprises vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof. In some cases, the therapeutic agent comprises vitamin C or its derivatives or pharmaceutically acceptable salts thereof. In some instances, the therapeutic agent is vitamin C. In some cases, vitamin C is L-ascorbic acid. In some cases, vitamin C is ascorbate.

In some instances, the therapeutic agent is a vitamin C derivative. In some instances, a derivative improves its solubility, absorption, biological half-life, and the like, or decreases the toxicity of the molecule, eliminate or attenuate any undesirable side effect of vitamin C. In some instances, a vitamin C derivative includes an isotopically labeled compound (e.g., with a radioisotope). In some instances, isotopes that are suitable for incorporation into vitamin C derivatives include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as, for example, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, and ³⁶Cl. In some instances, isotopically-labeled compounds, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays.

In some embodiments, a derivative of vitamin C is a deuterated version of the compound. In some instances, a deuterated version of the compound comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or more deuterium substitutions. In some cases, substitution with isotopes such as deuterium affords certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements.

In some cases, vitamin C derivatives comprise 6-O-palmitoyl L-ascorbic acid, ascorbyl palmitate, magnesium ascorbyl phosphate (MAP), ascorbyl tetra-isopalmitoyl (tetrahexyldecyl ascorbate), sodium ascorbyl phosphate (SAP), ascorbyl glucoside (ascorbic acid 2-glucoside), ethyl ascorbic acid, or L-ascorbyl stearate. In some cases, the vitamin C derivative is L-ascorbic acid 2-phosphate. In some instances, a vitamin C derivative further comprises a vitamin C derivative salt.

As used herein, a pharmaceutically acceptable salt or a derivative salt comprises a salt with an inorganic base, organic base, inorganic acid, organic acid, or basic or acidic amino acid. Salts of inorganic bases include, for example, alkali metals such as sodium or potassium; alkaline earth metals such as calcium and magnesium or aluminum; and ammonia. Salts of organic bases include, for example, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, and triethanolamine. Salts of inorganic acids include for example, hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid. Salts of organic acids include for example, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Salts of basic amino acids include, for example, arginine, lysine and ornithine. Acidic amino acids include, for example, aspartic acid and glutamic acid.

It should be understood that a reference to a pharmaceutically acceptable salt includes the solvent addition forms. In some embodiments, solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and are formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of compounds described herein are conveniently prepared or formed during the processes described herein. In addition, the compounds provided herein optionally exist in unsolvated as well as solvated forms.

In some instances, the therapeutic agent is a vitamin C analog. In some instances, a vitamin C analog refers to compounds that are structurally and functionally similar to, or mimics the effects of, vitamin C. In some instances, an analog mimics the biological effect of vitamin C. In other instances, an analog mimics the physical effect of vitamin C. In some cases, the vitamin C analog comprises 2-O-(beta-D-glucopyranosyl) ascorbic acid (AA-2βG).

In some instances, the therapeutic agent is a vitamin C metabolite. In some instances, a metabolite refers to the intermediates and products of vitamin C that is formed when vitamin C is metabolized. In additional embodiments, vitamin C is metabolized upon administration to an organism in need to produce a metabolite that is then used to produce a desired effect, including a desired therapeutic effect. In some instances, a metabolite of vitamin C is an active metabolite. The term “active metabolite” refers to a biologically active derivative of a compound that is formed when the compound is metabolized. The term “metabolized,” as used herein, refers to the sum of the processes (including, but not limited to, hydrolysis reactions and reactions catalyzed by enzymes) by which a particular substance is changed by an organism. Thus, in some instances, enzymes produce specific structural alterations to a compound. In some instances, a metabolite of vitamin C further enhances vitamin C uptake. In some instances, a vitamin C metabolite comprises L-threonic acid.

In some instances, the therapeutic agent is a vitamin C prodrug. In some instances, a prodrug has improved solubility in pharmaceutical compositions over the parent drug. In some embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of vitamin C. In some embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of vitamin C. In some instances, to produce a prodrug, a pharmaceutically active compound is modified such that the active compound will be regenerated upon in vivo administration. In some instances, the prodrug is designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. In some instances, prodrugs are designed as reversible drug derivatives, for use as modifiers to enhance drug transport to site-specific tissues. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound. (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392. Silverman (1992), The Organic Chemistry of Drug Design and Drug Action, Academic Press. Inc., San Diego, pages 352-401, Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985). In some instances, prodrugs of vitamin C comprise, for example, those described in PCT Publication No. WO2015048121.

In some instances, the therapeutic agent does not include an oxidized form of vitamin C. In some cases, the therapeutic agent does not include dehydroascorbic acid (DHA).

In some embodiments, administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof), induces an increase in the expression level of one or more epigenetic markers: ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, a marker selected from Table 1, or a combination thereof. In some instances, administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof), induces an increase in the expression level of one or more epigenetic markers: ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or a combination thereof. In some cases, administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof), induces an increase in the expression level of one or more epigenetic markers: ELOVL2, KLF14, PENK, or a combination thereof.

In some embodiments, an increase in the expression rate or level of ELOVL2, KLF14, PENK or a combination thereof further correlates to a decrease in cell senescence.

In some cases, an increase in the expression rate or level of ELOVL2, KLF14, PENK or a combination thereof further correlates to an increase in cell proliferation.

In some cases, an increase in the expression rate or level of ELOVL2, KLF14, PENK or a combination thereof further correlates to an increase in cell survival.

In additional cases, an increase in the expression rate or level of ELOVL2, KLF14, PENK, or a combination thereof further correlates to a decrease in DNA methylation. In some instances, an increase in the expression rate of ELOVL2, KLF14, PENK, or a combination thereof leads to a methylation pattern that mimics the methylation pattern of a sample obtained from a second subject. In some cases, the second subject is younger in chronological age relative to the first subject. In some cases, the second subject is younger in chronological age relative to the first subject by at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 50 years, or more.

In some cases, the first period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

In some embodiments, the method further comprises increasing the dose of the therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof) if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has not increased relative to the control. In some cases, the method comprises increasing the dose of the therapeutic agent if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased relative to the control and at a rate that is below a target range.

In other embodiments, the method further comprises decreasing or maintaining the dose of the therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof) if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased relative to the control. In some cases, the method comprises decreasing the dose of the therapeutic agent if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased relative to the control and at a rate that is above a target range. In other cases, the method comprises maintaining the dose of the therapeutic agent if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased relative to the control and at a rate that is within a target range.

In some instances, the dose of the therapeutic agent is increased, decreased, or maintained for a second period of time prior to redetermining the expression level of ELOVL2, KLF14, PENK, or a combination thereof. In some cases, the second period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

In some instances, the method further comprises administering to the first subject an additional therapeutic agent.

In some instances, the method further comprises administering a therapeutic agent to induce reprogramming of a cell into an induced pluripotent stem cell (iPSC). In some instances, the therapeutic agent is vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof. In some cases, the therapeutic agent comprises vitamin C or its derivatives or pharmaceutically acceptable salts thereof. In some instances, the therapeutic agent is vitamin C. In some cases, the therapeutic agent is L-ascorbic acid 2-phosphate.

In some embodiments, administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof), induces an increase in the expression level of a neurotrophin (e.g., BDNF NGF, or GDNF). In some cases, administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof), induces an increase in the expression level of BDNF. In some cases, administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof), induces an increase in the expression level of NGF. In some cases, administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof), induces an increase in the expression level of GDNF. In some cases, the therapeutic agent comprises vitamin C or its derivatives or pharmaceutically acceptable salts thereof. In some instances, the therapeutic agent is vitamin C. In some cases, the therapeutic agent is L-ascorbic acid 2-phosphate.

In some instances, an increase in the expression rate or level of a neurotrophin (e.g., BDNF, NGF, or GDNF) further correlates to a decrease in cell senescence. In some cases, an increase in the expression rate or level of BDNF further correlates to a decrease in cell senescence.

In some instances, an increase in the expression rate or level of a neurotrophin (e.g., BDNF, NGF, or GDNF) further correlates to an increase in cell proliferation. In some cases, an increase in the expression rate or level of BDNF further correlates to an increase in cell proliferation.

In some instances, an increase in the expression rate or level of a neurotrophin (e.g., BDNF, NGF, or GDNF) further correlates to an increase in cell survival. In some cases, an increase in the expression rate or level of BDNF further correlates to an increase in cell survival.

In some embodiments, the dose of a therapeutic agent is increased during the course of a treatment regimen if the expression rate or level of a neurotrophin (e.g., BDNF, NGF, or GDNF) is not increased relative to a control. In some cases, the dose of a therapeutic agent is increased during the course of a treatment regimen if the expression rate or level of a neurotrophin (e.g., BDNF NGF, or GDNF) is increased relative to a control but is at a rate that is below a target range.

In other embodiments, the dose of a therapeutic agent is decreased or maintained during the course of a treatment regimen if the expression rate or level of a neurotrophin (e.g., BDNF NGF or GDNF) has increased relative to a control. In such embodiments, the dose of a therapeutic agent is decreased or maintained during the course of a treatment regimen if the expression rate or level of a neurotrophin (e.g., BDNF, NGF, or GDNF) has increased relative to a control, but is at a rate that is above a target range.

In additional embodiments, the dose of a therapeutic agent is maintained during the course of a treatment regimen if the expression rate or level of a neurotrophin (e.g., BDNF NGF, or GDNF) has increased relative to a control. In such embodiments, the dose of a therapeutic agent is maintained during the course of a treatment regimen if the expression rate or level of a neurotrophin (e.g., BDNF NGF, or GDNF) has increased relative to a control, but is at a rate that is within a target range.

In some embodiments, administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof), induces an increase in the expression level of cortisol. In some cases, the therapeutic agent comprises vitamin C or its derivatives or pharmaceutically acceptable salts thereof. In some instances, the therapeutic agent is vitamin C. In some cases, the therapeutic agent is L-ascorbic acid 2-phosphate.

Methods in Reducing Methylation Level or Methylation Status

In some embodiments, disclosed herein is a method of retarding and/or reversing the biological age of a subject and the method comprises modulating the methylation pattern or level of one or more markers. In some instances, the one or more markers comprise ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, a neurotrophin (e.g., BDNF, NGF or GDNF), cortisol, or an epigenetic marker selected from Table 1. In some instances, the one or more markers comprise ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, a neurotrophin (e.g., BDNF, NGF or GDNF), cortisol, or a combination thereof. In some instances, the one or more markers comprise ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or a combination thereof. In some cases, the one or more markers comprise ELOVL2, KLF14, PENK, or a combination thereof.

In some embodiments, disclosed herein is a method of modulating the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof in a first subject, comprising (a) administering to the first subject a therapeutically effective dose of a therapeutic agent for a first time period; (b) obtaining a sample from the first subject; and (c) determining whether the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed in the first subject relative to a control by contacting the sample with a set of probes and detecting a set of hybridization products to determine the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof. In some instances, the methylation pattern of ELOVL2 is determined. In some instances, the methylation pattern of KLF14 is determined. In some instances, the methylation pattern of PENK is determined. In some cases, the methylation patterns of ELOVL2 and KLF14 are determined. In some cases, the methylation patterns of ELOVL2, KLF14, and PENK are determined.

In some instances, the therapeutic agent comprises vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof. In some cases, the therapeutic agent comprises vitamin C or its derivatives or pharmaceutically acceptable salts thereof. In some instances, the therapeutic agent is vitamin C. In some cases, vitamin C is L-ascorbic acid. In some cases, vitamin C is ascorbate.

In some instances, the therapeutic agent is a vitamin C derivative. In some cases, vitamin C derivatives comprise 6-O-palmitoyl L-ascorbic acid, ascorbyl palmitate, magnesium ascorbyl phosphate (MAP), ascorbyl tetra-isopalmitoyl (tetrahexyldecyl ascorbate), sodium ascorbyl phosphate (SAP), ascorbyl glucoside (ascorbic acid 2-glucoside), ethyl ascorbic acid, or L-ascorbyl stearate. In some cases, the vitamin C derivative is L-ascorbic acid 2-phosphate. In some instances, a vitamin C derivative further comprises a vitamin C derivative salt.

In some instances, the therapeutic agent is a vitamin C analog. In some cases, the vitamin C analog comprises 2-O-(beta-D-glucopyranosyl) ascorbic acid (AA-2βG).

In some instances, the therapeutic agent is a vitamin C metabolite. In some instances, a vitamin C metabolite comprises L-threonic acid.

In some instances, the therapeutic agent is a vitamin C prodrug. In some instances, prodrugs of vitamin C comprise, for example, those described in PCT Publication No. WO2015048121.

In some instances, the therapeutic agent does not include an oxidized form of vitamin C. In some cases, the therapeutic agent does not include dehydroascorbic acid (DHA).

In some embodiments, administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof), induces a decrease in the methylation status of one or more epigenetic markers: ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, a marker selected from Table 1, or a combination thereof. In some instances, administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof), induces a decrease in the methylation status of one or more epigenetic markers: ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or a combination thereof. In some cases, administration of a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof), induces a decrease in the methylation status of one or more epigenetic markers: ELOVL2, KLF14, PENK, or a combination thereof.

In some embodiments, a decrease in the methylation status of ELOVL2, KLF14, PENK, or a combination thereof further correlates to a decrease in cell senescence.

In some embodiments, a decrease in the methylation status of ELOVL2, KLF14, PENK, or a combination thereof further correlates to an increase in cell proliferation.

In some embodiments, a decrease in the methylation status of ELOVL2, KLF14, PENK, or a combination thereof further correlates to an increase in cell survival.

In some embodiments, a decrease in the methylation status of ELOVL2, KLF14, PENK, or a combination thereof leads to a methylation pattern that mimics the methylation pattern of a sample obtained from a second subject. In some cases, the second subject is younger in chronological age relative to the first subject. In some cases, the second subject is younger in chronological age relative to the first subject by at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 50 years, or more.

In some cases, the first period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

In some embodiments, the method further comprises increasing the dose of the therapeutic agent if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has not changed relative to the control. In some cases, the method comprises increasing the dose of the therapeutic agent if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control and to a degree lower than a target range.

In other embodiments, the method further comprises decreasing or maintaining the dose of the therapeutic agent if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control. In some cases, the method comprises decreasing the dose of the therapeutic agent if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control and to a degree above a target range. In additional cases, the method comprises maintaining the dose of the therapeutic agent if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control and to a degree within a target range.

In some instances, the dose of the therapeutic agent is increased, decreased, or maintained for a second period of time prior to redetermining the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof.

In some cases, the second period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

In some instances, the method further comprises administering to the first subject an additional therapeutic agent.

Control

Various methodologies described herein include a step that involves comparing a value, level, feature, characteristic, property, etc. to a suitable control, referred to interchangeably herein as an appropriate control, a control sample, or as a control. In some embodiments, a control is a value, level, feature, characteristic, property, etc., determined in a cell, a tissue, an organ, or a sample obtained from a patient. In some instances, the cell, tissue, organ, or sample is a young cell, tissue, organ, or sample. In some cases, the cell tissue, organ, or sample is an aged cell, tissue, organ, or sample. In some instances, the cell, tissue, organ, or sample is obtained from an individual with a chronological age of less than 1, 2, 3, 4, 5, 10, 12, 14, 15, 18, 20, 25, 30, 35, 40, 45, or 50 years. In some instances, the cell, tissue, organ, or sample is obtained from an individual with a chronological age of more than 1, 2, 3, 4, 5, 10, 12, 14, 15, 18, 20, 25, 30, 35, 40, 45, or 50 years.

In some cases, the control comprises the expression level of ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, a neurotrophin (e.g., BDNF NGF or GDNF), cortisol, an epigenetic marker selected from Table 1, or a combination thereof obtained from a sample from the subject prior to administration of the therapeutic agent. In some cases, the control comprises the expression level of ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, a neurotrophin (e.g., BDNF, NGF or GDNF), cortisol, or a combination thereof obtained from a sample from the subject prior to administration of the therapeutic agent. In some cases, the control comprises the expression level of ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or a combination thereof obtained from a sample from the subject prior to administration of the therapeutic agent. In some cases, the control comprises the expression level of ELOVL2, KLF14, PENK, or a combination thereof obtained from a sample from the subject prior to administration of the therapeutic agent.

In some cases, the control comprises a normalized expression level of ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, a neurotrophin (e.g., BDNF, NGF or GDNF), cortisol, an epigenetic marker selected from Table 1, or a combination thereof obtained from a set of samples without exposure to the therapeutic agent. In some cases, the control comprises a normalized expression level of ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, a neurotrophin (e.g., BDNF, NGF or GDNF), cortisol, or a combination thereof obtained from a set of samples without exposure to the therapeutic agent. In some cases, the control comprises a normalized expression level of ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or a combination thereof obtained from a set of samples without exposure to the therapeutic agent. In some cases, the control comprises a normalized expression level of ELOVL2, KLF14, PENK, or a combination thereof obtained from a set of samples without exposure to the therapeutic agent. In some cases, the set of samples are a set of cell samples.

In some cases, the control comprises the methylation pattern of ELOV2, KLF14, PENK, FHL2, SMC4, SLC125A, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, an epigenetic marker selected from Table 1, or a combination thereof obtained from a sample from the subject prior to administration of the therapeutic agent. In some cases, the control comprises the methylation pattern of ELOVL2, KLF14, PENK, FHL2, SMC4, SLC1245, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or a combination thereof obtained from a sample from the subject prior to administration of the therapeutic agent. In some cases, the control comprises the methylation pattern of ELOVL2, KLF14, PENK or a combination thereof obtained from a sample from the subject prior to administration of the therapeutic agent.

In some cases, the control comprises a normalized methylation pattern of ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, an epigenetic marker selected from Table 1, or a combination thereof obtained from a set of samples without exposure to the therapeutic agent. In some cases, the control comprises a normalized methylation pattern of ELOVL2, KLF14, PENK, FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, or a combination thereof obtained from a set of samples without exposure to the therapeutic agent. In some cases, the control comprises a normalized methylation pattern of ELOVL2, KLF14, PENK or a combination thereof obtained from a set of samples without exposure to the therapeutic agent. In some cases, the set of samples are a set of cell samples.

In some instances, a control is a positive control, e.g., a methylation profile obtained from a sample of an aged individual, or is a negative control, e.g., a methylation profile obtained from a sample of a young individual. In some instances, a control is also referred to as a training set or training dataset.

Diseases or Indications

In some embodiments, one or more samples are obtained from a subject having a disease or indication. In some instances, the disease or condition is an aging-related disease or condition. In some instances, the disease or indication is a metabolic disease or condition. In some instances, the disease or indication is an ELOVL2-associated disease or indication, a KLF14-associated disease or indication, or a PENK-associated disease or indication. In some cases, the disease or indication is Werner syndrome, progeria, or post-traumatic stress disorder.

In some embodiments, also disclosed herein is a method of increasing the expression level of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having a disease or indication by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the expression level of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been elevated. In some embodiments, further described herein is a method of modulating the methylation pattern of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having a disease or indication by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the methylation pattern of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been changed. In some instances, the disease or indication is a metabolic disease or condition. In some instances, the disease or indication is an ELOVL2-associated disease or indication, a KLF14-associated disease or indication, or a PENK-associated disease or indication. In some cases, the disease or indication is Werner syndrome, progeria, or post-traumatic stress disorder.

Diabetes

In some embodiments, a metabolic disease or condition is diabetes (diabetes mellitus, DM). In some instances, diabetes is type 1 diabetes, type 2 diabetes, type 3 diabetes, type 4 diabetes, double diabetes, latent autoimmune diabetes (LAD), gestational diabetes, neonatal diabetes mellitus (NDM), maturity onset diabetes of the young (MODY), Wolfram syndrome, Alström syndrome, prediabetes, or diabetes insipidus. Type 2 diabetes, also called non-insulin dependent diabetes, is the most common type of diabetes accounting for 95% of all diabetes cases. In some instances, type 2 diabetes is caused by a combination of factors, including insulin resistance due to pancreatic beta cell dysfunction, which in turn leads to high blood glucose levels. In some cases, increased glucagon levels stimulate the liver to produce an abnormal amount of unneeded glucose, which contributes to high blood glucose levels.

Type 1 diabetes, also called insulin-dependent diabetes, comprises about 5% to 10% of all diabetes cases. Type 1 diabetes is an autoimmune disease where T cells attack and destroy insulin-producing beta cells in the pancreas. In some embodiments, Type 1 diabetes is caused by genetic and environmental factors.

In some embodiments, the term double diabetes is used to describe patients diagnosed with both type 1 and 2 diabetes.

Type 4 diabetes is a recently discovered type of diabetes affecting about 20% of diabetic patients age 65 and over. In some embodiments, type 4 diabetes is characterized by age-associated insulin resistance.

In some embodiments, type 3 diabetes is used as a term for Alzheimer's disease resulting in insulin resistance in the brain.

LAD, also known as slow onset type 1 diabetes, is a slow developing form of type 1 diabetes where diagnosis frequently occurs after age 30. In some embodiments, LAD is further classified into latent autoimmune diabetes in adults (LADA) or latent autoimmune diabetes in the young (LADY) or latent autoimmune diabetes in children (LADC).

Prediabetes, also known as borderline diabetes, is a precursor stage to diabetes mellitus. In some cases, prediabetes is characterized by abnormal OGTT, fasting plasma glucose test, and hemoglobin A1C test results. In some embodiments, prediabetes is further classified into impaired fasting glycaemia or impaired fasting glucose (IFG) and impaired glucose tolerance (IGT). IFG is a condition in which blood glucose levels are higher than normal levels, but not elevated enough to be diagnosed as diabetes mellitus. IGT is a pre-diabetic state of abnormal blood glucose levels associated with insulin resistance and increased risk of cardiovascular pathology.

In some embodiments, the sample is obtained from a subject having diabetes. In some instances, the sample is obtained from a subject having type 1 diabetes, type 2 diabetes, type 3 diabetes, type 4 diabetes, double diabetes, latent autoimmune diabetes (LAD), gestational diabetes, neonatal diabetes mellitus (NDM), maturity onset diabetes of the young (MODY), Wolfram syndrome, Alström syndrome, prediabetes, or diabetes insipidus. In some cases, the sample is obtained from a subject having type 1 diabetes. In other cases, the sample is obtained from a subject having type 2 diabetes. In additional cases, the sample is obtained from a subject having prediabetes.

In some embodiments, also disclosed herein is a method of increasing the expression level of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having diabetes by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the expression level of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been elevated. In some embodiments, further described herein is a method of modulating the methylation pattern of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having diabetes by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the methylation pattern of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been changed.

In some embodiments, the sample is obtained from a subject having an elevated body mass index (BMI). In some instances, the elevated BMI is from about 25 kg/m² to about 40 kg/m². In some instances, the elevated BMI is from about 25 kg/m² to about 29.9 kg/m², from about 30 kg/m² to about 34.9 kg/m², or from about 35 kg/m² to about 39 kg/m². In some cases, the elevated BMI is a BMI of 25 kg/m², 26 kg/m², 27 kg/m², 28 kg/m², 29 kg/m², 30 kg/m², 35 kg/m², 40 kg/m² or more.

In some embodiments, also disclosed herein is a method of increasing the expression level of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having an elevated BMI by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the expression level of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been elevated. In some embodiments, further described herein is a method of modulating the methylation pattern of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having an elevated BMI by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the methylation pattern of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been changed.

Werner Syndrome

In some embodiments, the sample is obtained from a subject having Werner syndrome. Werner syndrome (also known as adult progeria or WS) is an autosomal recessive progeroid syndrome with phenotype of premature aging. In some instances, patient with Werner syndrome is characterized with growth retardation, short stature, premature graying of hair, alopecia (hair loss), wrinkling, prematurely aged faces with beaked noses, skin atrophy (wasting away) with scleroderma-like lesions, lipodystrophy (loss of fat tissues), abnormal fat deposition leading to thin legs and arms, and/or severe ulcerations around the Achilles tendon and malleoli (around ankles).

In some instances, Werner syndrome is caused by mutations in the WRN (Werner Syndrome, RecQ helicase-like) gene which encodes a 1432 amino acid protein, WRNp protein, which is involved in DNA repair and replication. In some instances, a patient with Werner syndrome losses the activity of WRNp protein, and further exhibits accelerated telomere shortening and telomere dysfunction.

In some embodiments, also disclosed herein is a method of increasing the expression level of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having Werner syndrome by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the expression level of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been elevated. In some embodiments, further described herein is a method of modulating the methylation pattern of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having Werner syndrome by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the methylation pattern of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been changed.

Progeria

In some embodiments, the sample is obtained from a subject having progeria. Progeria (or Hutchinson-Gilford progeria syndrome, HGPS, or progeria syndrome) is a rare genetic disorder in which the symptoms resemble premature aging. In some instances, progeria is manifested at a young age. In some instances, the first sign of symptoms occurs during the first few months of infancy and include a failure to thrive and a localized scleroderma-like skin condition. In some instances, secondary conditions occur around 18-24 months and include alopecia and a distinctive physical appearance (e.g., a small face with a shallow recessed jaw and/or a pinched nose). In some cases, additional symptoms include wrinkled skin, atherosclerosis, kidney failure, loss of eyesight, and/or cardiovascular disorders.

In some instances, progeria is caused by a cytosine to thymine mutation at position 1824 of the LMNA gene. In some cases, the mutation induces a 5′ cryptic splice site which then leads to the production of a prelamin A protein variant. The preliamin A protein variant subsequently induces an abnormally shaped nucleus and impedes cell division, leading to progeria.

In some embodiments, also disclosed herein is a method of increasing the expression level of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having progeria by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the expression level of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been elevated. In some embodiments, further described herein is a method of modulating the methylation pattern of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having progeria by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the methylation pattern of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been changed.

Post-Traumatic Stress Disorder

In some embodiments, the sample is obtained from a subject having post-traumatic stress disorder (PTSD). Post-traumatic stress disorder (PTSD) is a metal disorder developed after experiencing a traumatic event. In some embodiments, also disclosed herein is a method of increasing the expression level of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having PTSD by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the expression level of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been elevated. In some embodiments, further described herein is a method of modulating the methylation pattern of an epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) in a subject having PTSD by administering to the subject a therapeutically effective dose of a therapeutic agent and determining whether the methylation pattern of the epigenetic marker (e.g., ELOVL2, KLF14, PENK, or a combination thereof) has been changed.

Pharmaceutical Compositions and Formulations

In some embodiments, the pharmaceutical composition and formulations comprising a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof) are administered to a subject by multiple administration routes, including but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal administration routes. In some instances, the pharmaceutical composition comprising a therapeutic agent (e.g., vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof) is formulated for oral administration.

In some embodiments, the pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.

In some embodiments, the pharmaceutical formulations include a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carragcenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975. Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980, and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

In some instances, the pharmaceutical formulations further include pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric, and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases, and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

In some instances, the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions. Suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

In some embodiments, the pharmaceutical formulations include, but are not limited to, sugars like trehalose, sucrose, mannitol, maltose, and glucose, or salts like potassium phosphate, sodium citrate, ammonium sulfate and/or other agents such as heparin to increase the solubility and in vivo stability of polypeptides.

In some instances, the pharmaceutical formulations further include diluent which are used to stabilize compounds because they provide a more stable environment. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain instances, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling. Such compounds can include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®, dibasic calcium phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugar, such as Di-Pac® (Amstar), mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, and the like.

In some cases, the pharmaceutical formulations include disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance. The term “disintegrate” include both the dissolution and dispersion of the dosage form when contacted with gastrointestinal fluid. Examples of disintegration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum® HV (magnesium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.

In some instances, the pharmaceutical formulations include filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein for preventing, reducing or inhibiting adhesion or friction of materials. Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starch such as corn starch, silicone oil, a surfactant, and the like.

Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers can also function as dispersing agents or wetting agents.

Solubilizers include compounds such as triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like.

Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like. Exemplary stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium biphosphate dehydrate, propylene glycol, metacresol or m-cresol, zinc acetate, polysorbate-20 or Tween® 20, or trometamol.

Suspending agents include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40, Sometimes, surfactants is included to enhance physical stability or for other purposes.

Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.

Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.

Therapeutic Regimens

In some embodiments, a therapeutic agent described herein is administered for one or more times a day. In some embodiments, a therapeutic agent described herein is administered once per day, twice per day, three times per day or more. In some cases, a therapeutic agent described herein is administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or more. In some cases, a therapeutic agent described herein is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

In some instances, a therapeutic agent described herein is administered at a dose range of from about 0.1 μg/mL to about 200 μg/mL. In some instances, the therapeutic agent described herein is administered at a dose range of from about 1 μg/mL to about 150 μg/mL, from about 5 μg/mL to about 100 μg/mL, from about 10 μg/mL to about 100 μg/mL, from about 20 μg/mL to about 100 μg/mL, from about 30 μg/mL to about 100 μg/mL, from about 50 μg/mL to about 100 μg/mL, from about 1 μg/mL to about 50 μg/mL, from about 5 μg/mL to about 50 μg/mL, from about 10 μg/mL to about 50 μg/mL, from about 20 μg/mL to about 50 μg/mL, from about 30 μg/mL to about 50 μg/mL, from about 50 μg/mL to about 200 μg/mL, from about 80 μg/mL to about 200 μg/mL, from about 100 μg/mL to about 200 μg/mL, or from about 150 μg/mL to about 200 μg/mL.

In some instances, the therapeutic agent described herein is administered at a dose of about 0.1 μg/mL, 1 μg/mL, 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 25 μg/mL, 30 μg/mL, 35 μg/mL, 40 μg/mL, 45 μg/mL, 50 μg/mL, 55 g/mL, 60 μg/mL, 65 μg/mL, 70 μg/mL, 75 μg/mL, 80 μg/mL, 85 μg/mL, 90 μg/mL, 95 μg/mL, 100 μg/mL, 110 μg/mL, 120 μg/mL, 130 μg/mL, 140 μg/mL, 150 μg/mL, 160 μg/mL, 170 μg/mL, 180 μg/mL, 190 μg/mL, or about 200 μg/mL.

In some instances, the therapeutic agent is vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof. In some instances, vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof is administered at a dose range of from about 0.1 μg/mL to about 200 μg/mL. In some instances, vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof is administered at a dose range of from about 1 μg/mL to about 150 μg/mL, from about 5 μg/mL to about 100 μg/mL, from about 10 μg/mL to about 100 μg/mL, from about 20 μg/mL to about 100 μg/mL, from about 30 g/mL to about 100 μg/mL, from about 50 μg/mL to about 100 μg/mL, from about 1 μg/mL to about 501 μg/mL, from about 5 μg/mL to about 50 g/mL, from about 10 μg/mL to about 50 μg/mL, from about 20 μg/mL to about 50 μg/mL, from about 30 μg/mL to about 50 μg/mL, from about 50 μg/mL to about 200 μg/mL, from about 80 μg/mL to about 200 μg/mL, from about 100 μg/mL to about 200 μg/mL, or from about 150 μg/mL to about 200 μg/mL.

In some instances, vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof is administered at a dose of about 0.1 μg/mL, 1 μg/mL, 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 25 μg/mL, 30 μg/mL, 35 μg/mL, 40 μg/mL, 45 μg/mL, 50 μg/mL, 55 μg/mL, 60 μg/mL, 65 μg/mL, 70 μg/mL, 75 μg/mL, 80 μg/mL, 85 μg/mL, 90 μg/mL, 95 μg/mL, 100 μg/mL, 110 μg/mL, 120 μg/mL, 130 μg/mL, 140 μg/mL, 150 μg/mL, 160 μg/mL, 170 μg/mL, 180 μg/mL, 190 μg/mL, or about 200 μg/mL.

In some embodiments, a dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof greater than 200 μg/mL increases reactive oxidative species. In some cases, a dose of vitamin C or its derivatives, analogs, metabolites, prodrugs, or pharmaceutically acceptable salts thereof greater than 200 μg/mL leads to a methylation pattern that mimics the methylation pattern of a sample obtained from a third subject who is older in chronological age relative to the first subject. In some instances, the third subject is older in chronological age relative to the first subject by at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 50 years, or more.

The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages is altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

In some embodiments, toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50. Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.

Detection Methods

In some embodiments, a number of methods are utilized to measure, detect, determine, identify, and characterize the expression level and the methylation status/level of a gene or a epigenetic marker (i.e., a region/fragment of DNA or a region/fragment of genome DNA (e.g., CpG island-containing region/fragment)) in determining the biological age of a subject and the progression or regression of the biological age of the subject in the presence of a therapeutic agent.

In some instances, the expression level and/or the methylation profile is generated from a biological sample isolated from an individual. In some embodiments, the biological sample is a biopsy. In some instances, the biological sample is a tissue sample. In other instances, the biological sample is a cell-free biological sample. In other instances, the biological sample is a circulating tumor DNA sample. In one embodiment, the biological sample is a cell free biological sample containing circulating tumor DNA.

In some embodiments, an epigenetic marker (also referred herein as a marker) is obtained from a tissue sample. In some instances, a tissue corresponds to any cell(s). Different types of tissue correspond to different types of cells (e.g., liver, lung, blood, connective tissue, and the like), but also healthy cells vs. tumor cells or to tumor cells at various stages of neoplasia, or to displaced malignant tumor cells. In some embodiments, a tissue sample further encompasses a clinical sample, and also includes cells in culture, cell supernatants, organs, and the like. Samples also comprise fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or study by immunohistochemistry.

In some embodiments, an epigenetic marker is obtained from a liquid sample. In some embodiments, the liquid sample comprises blood and other liquid samples of biological origin (including, but not limited to, peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing, svnovial fluid, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood. In some embodiments, the biological fluid is blood, a blood derivative or a blood fraction, e.g., serum or plasma. In a specific embodiment, a sample comprises a blood sample. In another embodiment, a serum sample is used. In another embodiment, a sample comprises urine. In some embodiments, the liquid sample also encompasses a sample that has been manipulated in any way after their procurement, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washed, or enriched for certain cell populations.

In some embodiments, an epigenetic marker is methylated or unmethylated in a normal sample (e.g., normal or control tissue without disease, or normal or control body fluid, stool, blood, serum, amniotic fluid), most importantly in healthy stool, blood, serum, amniotic fluid or other body fluid. In other embodiments, an epigenetic marker is hypomethylated or hypermethylated in a sample from a patient having or at risk of a disease (e.g., one or more indications described herein); for example, at a decreased or increased (respectively) methylation frequency of at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% in comparison to a normal sample. In one embodiment, a sample is also hypomethylated or hypermethylated in comparison to a previously obtained sample analysis of the same patient having or at risk of a disease (e.g., one or more indications described herein), particularly to compare progression of a disease.

In some embodiments, a methylome comprises a set of epigenetic markers, such as an epigenetic marker described above. In some instances, a methylome that corresponds to the methylome of a tumor of an organism (e.g., a human) is classified as a tumor methylome. In some cases, a tumor methylome is determined using tumor tissue or cell-free (or protein-free) tumor DNA in a biological sample. Other examples of methylomes of interest include the methylomes of organs that contribute DNA into a bodily fluid (e.g. methylomes of tissue such as brain, breast, lung, the prostrate and the kidneys, plasma, etc.).

In some embodiments, a plasma methylome is the methylome determined from the plasma or serum of an animal (e.g., a human). In some instances, the plasma methylome is an example of a cell-free or protein-free methylome since plasma and serum include cell-free DNA. The plasma methylome is also an example of a mixed methylome since it is a mixture of tumor and other methylomes of interest. In some instances, the urine methylome is determined from the urine sample of a subject. In some cases, a cellular methylome corresponds to the methylome determined from cells (e.g., tissue cells from an organ such as brain, lung, breast and the like) of the patient. The methylome of the blood cells is called the blood cell methylome (or blood methylome).

In some embodiments, DNA (e.g., genomic DNA such as extracted genomic DNA or treated genomic DNA) is isolated by any means standard in the art, including the use of commercially available kits. Briefly, wherein the DNA of interest is encapsulated in by a cellular membrane the biological sample is disrupted and lysed by enzymatic, chemical or mechanical means. In some cases, the DNA solution is then cleared of proteins and other contaminants e.g. by digestion with proteinase K. The DNA is then recovered from the solution. In such cases, this is carried out by means of a variety of methods including salting out, organic extraction or binding of the DNA to a solid phase support. In some instances, the choice of method is affected by several factors including time, expense and required quantity of DNA.

Wherein the sample DNA is not enclosed in a membrane (e.g. circulating DNA from a cell free sample such as blood or urine) methods standard in the art for the isolation and/or purification of DNA are optionally employed (See, for example, Bettegowda et al. Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies. Sci. Transl. Med, 6(224): ra24. 2014). Such methods include the use of a protein degenerating reagent e.g. chaotropic salt e.g. guanidine hydrochloride or urea; or a detergent e.g. sodium dodecyl sulphate (SDS), cyanogen bromide. Alternative methods include but are not limited to ethanol precipitation or propanol precipitation, vacuum concentration amongst others by means of a centrifuge. In some cases, the person skilled in the art also make use of devices such as filter devices e.g. ultrafiltration, silica surfaces or membranes, magnetic particles, polystyrol particles, polystyrol surfaces, positively charged surfaces, and positively charged membranes, charged membranes, charged surfaces, charged switch membranes, charged switched surfaces.

In some instances, once the nucleic acids have been extracted, methylation analysis is carried out by any means known in the art. A variety of methylation analysis procedures are known in the art and may be used to practice the methods disclosed herein. These assays allow for determination of the methylation state of one or a plurality of CpG sites within a tissue sample. In addition, these methods may be used for absolute or relative quantification of methylated nucleic acids. Such methylation assays involve, among other techniques, two major steps. The first step is a methylation specific reaction or separation, such as (i) bisulfite treatment, (ii) methylation specific binding, or (iii) methylation specific restriction enzymes. The second major step involves (i) amplification and detection, or (ii) direct detection, by a variety of methods such as (a) PCR (sequence-specific amplification) such as Taqman®, (b) DNA sequencing of untreated and bisulfite-treated DNA, (c) sequencing by ligation of dye-modified probes (including cyclic ligation and cleavage), (d) pyrosequencing, (e) single-molecule sequencing. (f) mass spectroscopy, or (g) Southern blot analysis.

Additionally, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA may be used, e.g., the method described by Sadri and Hornsby (1996. Nucl. Acids Res. 24:5058-5059), or COBRA (Combined Bisulfite Restriction Analysis) (Xiong and Laird, 1997, Nucleic Acids Res. 25:2532-2534). COBRA analysis is a quantitative methylation assay useful for determining DNA methylation levels at specific gene loci in small amounts of genomic DNA. Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA. Methylation-dependent sequence differences are first introduced into the genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Frommer et al, 1992, Proc. Nat. Acad. Sci. USA, 89, 1827-1831). PCR amplification of the bisulfite converted DNA is then performed using primers specific for the CpG sites of interest, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specific, labeled hybridization probes. Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels. In addition, this technique can be reliably applied to DNA obtained from micro-dissected paraffin-embedded tissue samples. Typical reagents (e.g., as might be found in a typical COBRA-based kit) for COBRA analysis may include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); restriction enzyme and appropriate buffer; gene-hybridization oligo; control hybridization oligo; kinase labeling kit for oligo probe; and radioactive nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffer; sulfo nation buffer; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity column), desulfonation buffer; and DNA recovery components.

In an embodiment, the methylation profile of selected CpG sites is determined using methylation-Specific PCR (MSP). MSP allows for assessing the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes (Herman et al. 1996, Proc. Nat. Acad. Sci. USA, 93, 9821-9826; U.S. Pat. Nos. 5,786,146, 6,017,704, 6,200,756, 6,265,171 (Herman and Baylin); U.S. Pat. Pub. No. 2010/0144836 (Van Engeland et al)). Briefly, DNA is modified by a deaminating agent such as sodium bisulfite to convert unmethylated, but not methylated cytosines to uracil, and subsequently amplified with primers specific for methylated versus unmethylated DNA. In some instances, typical reagents (e.g., as might be found in a typical MSP-based kit) for MSP analysis include, but are not limited to: methylated and unmethylated PCR primers for specific gene (or methylation-altered DNA sequence or CpG island), optimized PCR buffers and deoxynucleotides, and specific probes. The ColoSure™ test is a commercially available test for colon cancer based on the MSP technology and measurement of methylation of the vimentin gene (Itzkowitz et al, 2007, Clin Gastroenterol. Hepatol. 5(1), 111-117). Alternatively, one may use quantitative multiplexed methylation specific PCR (QM-PCR), as described by Fackler et al. Fackler et al, 2004, Cancer Res. 64(13) 4442-4452; or Fackler et al, 2006, Clin. Cancer Res. 12(11 Pt 1) 3306-3310.

In an embodiment, the methylation profile of selected CpG sites is determined using MethyLight and/or Heavy Methyl Methods. The MethyLight and Heavy Methyl assays are a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (Taq Man®) technology that requires no further manipulations after the PCR step (Eads, C. A. et al, 2000, Nucleic Acid Res. 28, e 32; Cottrell et al, 2007, J. Urology 177, 1753, U.S. Pat. No. 6,331,393 (Laird et al)). Briefly, the MethyLight process begins with a mixed sample of genomic DNA that is converted, in a sodium bisulfite reaction, to a mixed pool of methylation-dependent sequence differences according to standard procedures (the bisulfite process converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed either in an “unbiased” (with primers that do not overlap known CpG methylation sites) PCR reaction, or in a “biased” (with PCR primers that overlap known CpG dinucleotides) reaction. In some cases, sequence discrimination occurs either at the level of the amplification process or at the level of the fluorescence detection process, or both. In some cases, the MethyLight assay is used as a quantitative test for methylation patterns in the genomic DNA sample, wherein sequence discrimination occurs at the level of probe hybridization. In this quantitative version, the PCR reaction provides for unbiased amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe overlie any CpG dinucleotides. Alternatively, a qualitative test for genomic methylation is achieved by probing of the biased PCR pool with either control oligonucleotides that do not “cover” known methylation sites (a fluorescence-based version of the “MSP” technique), or with oligonucleotides covering potential methylation sites. Typical reagents (e.g., as might be found in a typical MethyLight-based kit) for MethyLight analysis may include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); TaqMan® probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase. The MethyLight technology is used for the commercially available tests for lung cancer (epi proLung BL Reflex Assay); colon cancer (epi proColon assay and mSEPT9 assay) (Epigenomics, Berlin, Germany) PCT Pub. No. WO 2003/064701 (Schweikhardt and Sledziewski).

Quantitative MethyLight uses bisulfite to convert genomic DNA and the methylated sites are amplified using PCR with methylation independent primers. Detection probes specific for the methylated and unmethylated sites with two different fluorophores provides simultaneous quantitative measurement of the methylation. The Heavy Methyl technique begins with bisulfate conversion of DNA. Next specific blockers prevent the amplification of unmethylated DNA. Methylated genomic DNA does not bind the blockers and their sequences will be amplified. The amplified sequences are detected with a methylation specific probe. (Cottrell et al. 2004, Nuc. Acids Res. 32:e10).

The Ms-SNuPE technique is a quantitative method for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA, followed by single-nucleotide primer extension (Gonzalgo and Jones, 1997, Nucleic Acids Res. 25, 2529-2531). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site(s) of interest. In some cases, small amounts of DNA are analyzed (e.g., micro-dissected pathology sections), and the method avoids utilization of restriction enzymes for determining the methylation status at CpG sites. Typical reagents (e.g., as is found in a typical Ms-SNuPE-based kit) for Ms-SNuPE analysis include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island), optimized PCR buffers and deoxynucleotides; gel extraction kit; positive control primers; Ms-SNuPE primers for specific gene; reaction buffer (for the Ms-SNuPE reaction); and radioactive nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery regents or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components.

In another embodiment, the methylation status of selected CpG sites is determined using differential Binding-based Methylation Detection Methods. For identification of differentially methylated regions, one approach is to capture methylated DNA. This approach uses a protein, in which the methyl binding domain of MBD2 is fused to the Fc fragment of an antibody (MBD-FC) (Gebhard et al, 2006, Cancer Res. 66:6118-6128; and PCT Pub. No. WO 2006/056480 A2 (Relhi)). This fusion protein has several advantages over conventional methylation specific antibodies. The MBD FC has a higher affinity to methylated DNA and it binds double stranded DNA. Most importantly the two proteins differ in the way they bind DNA. Methylation specific antibodies bind DNA stochastically, which means that only a binary answer can be obtained. The methyl binding domain of MBD-FC, on the other hand, binds DNA molecules regardless of their methylation status. The strength of this protein-DNA interaction is defined by the level of DNA methylation. After binding genomic DNA, eluate solutions of increasing salt concentrations can be used to fractionate non-methylated and methylated DNA allowing for a more controlled separation (Gebhard et al, 2006, Nucleic Acids Res. 34: e82). Consequently this method, called Methyl-CpG immunoprecipitation (MCIP), not only enriches, but also fractionates genomic DNA according to methylation level, which is particularly helpful when the unmethylated DNA fraction should be investigated as well.

In an alternative embodiment, a 5-methyl cytidine antibody to bind and precipitate methylated DNA. Antibodies are available from Abeam (Cambridge, Mass.), Diagenode (Sparta, N.J.) or Eurogentec (c/o AnaSpec, Fremont, Calif.). Once the methylated fragments have been separated they may be sequenced using microarray based techniques such as methylated CpG-island recovery assay (MIRA) or methylated DNA immunoprecipitation (MeDIP) (Pelizzola et al, 2008, Genome Res. 18, 1652-1659; O'Geen et al, 2006, BioTechniques 41(5), 577-580, Weber et al, 2005, Nat. Genet. 37, 853-862; Horak and Snyder, 2002, Methods Enzymol, 350, 469-83; Lieb, 2003, Methods Mol Biol, 224, 99-109). Another technique is methyl-CpG binding domain column/segregation of partly melted molecules (MBD/SPM, Shiraishi et al, 1999, Proc. Natl. Acad. Sci. USA 96(6):2913-2918).

In some embodiments, methods for detecting methylation include randomly shearing or randomly fragmenting the genomic DNA, cutting the DNA with a methylation-dependent or methylation-sensitive restriction enzyme and subsequently selectively identifying and/or analyzing the cut or uncut DNA. Selective identification can include, for example, separating cut and uncut DNA (e.g., by size) and quantifying a sequence of interest that was cut or, alternatively, that was not cut. See, e.g., U.S. Pat. No. 7,186,512. Alternatively, the method can encompass amplifying intact DNA after restriction enzyme digestion, thereby only amplifying DNA that was not cleaved by the restriction enzyme in the area amplified. See, e.g., U.S. Pat. Nos. 7,910,296; 7,901,880; and 7,459,274. In some embodiments, amplification can be performed using primers that are gene specific.

For example, there are methyl-sensitive enzymes that preferentially or substantially cleave or digest at their DNA recognition sequence if it is non-methylated. Thus, an unmethylated DNA sample is cut into smaller fragments than a methylated DNA sample. Similarly, a hypermethylated DNA sample is not cleaved. In contrast, there are methyl-sensitive enzymes that cleave at their DNA recognition sequence only if it is methylated. Methyl-sensitive enzymes that digest unmethylated DNA suitable for use in methods of the technology include, but are not limited to, Hpall, Hhal, Maell, BstUI and Acil. In some instances, an enzyme that is used is Hpall that cuts only the unmethylated sequence CCGG. In other instances, another enzyme that is used is Hhal that cuts only the unmethylated sequence GCGC. Both enzymes are available from New England BioLabs®, Inc. Combinations of two or more methyl-sensitive enzymes that digest only unmethylated DNA are also used. Suitable enzymes that digest only methylated DNA include, but are not limited to, Dpnl, which only cuts at fully methylated 5′-GATC sequences, and McrBC, an endonuclease, which cuts DNA containing modified cytosines (5-methylcytosine or 5-hydroxymethylcytosine or N4-methylcytosine) and cuts at recognition site 5′ . . . PumC(N4o-3ooo) PumC . . . 3′ (New England BioLabs, Inc., Beverly, Mass.). Cleavage methods and procedures for selected restriction enzymes for cutting DNA at specific sites are well known to the skilled artisan. For example, many suppliers of restriction enzymes provide information on conditions and types of DNA sequences cut by specific restriction enzymes, including New England BioLabs, Pro-Mega Biochems, Boehringer-Mannheim, and the like. Sambrook et al. (See Sambrook et al. Molecular Biology: A Laboratory Approach, Cold Spring Harbor, N.Y. 1989) provide a general description of methods for using restriction enzymes and other enzymes.

In some instances, a methylation-dependent restriction enzyme is a restriction enzyme that cleaves or digests DNA at or in proximity to a methylated recognition sequence, but does not cleave DNA at or near the same sequence when the recognition sequence is not methylated. Methylation-dependent restriction enzymes include those that cut at a methylated recognition sequence (e.g., Dpnl) and enzymes that cut at a sequence near but not at the recognition sequence (e.g., McrBC). For example, McrBC's recognition sequence is 5′ RmC (N40-3000) RmC 3′ where “R” is a purine and “mC” is a methylated cytosine and “N40-3000” indicates the distance between the two RmC half sites for which a restriction event has been observed. McrBC generally cuts close to one half-site or the other, but cleavage positions are typically distributed over several base pairs, approximately 30 base pairs from the methylated base. McrBC sometimes cuts 3′ of both half sites, sometimes 5′ of both half sites, and sometimes between the two sites. Exemplary methylation-dependent restriction enzymes include, e.g., McrBC, McrA, MrrA, Bisl, Glal and Dpnl. One of skill in the art will appreciate that any methylation-dependent restriction enzyme, including homologs and orthologs of the restriction enzymes described herein, is also suitable for use with one or more methods described herein.

In some cases, a methylation-sensitive restriction enzyme is a restriction enzyme that cleaves DNA at or in proximity to an unmethylated recognition sequence but does not cleave at or in proximity to the same sequence when the recognition sequence is methylated. Exemplary methylation-sensitive restriction enzymes are described in, e.g., McClelland et al, 22(17) NUCLEIC ACIDS RES. 3640-59 (1994). Suitable methylation-sensitive restriction enzymes that do not cleave DNA at or near their recognition sequence when a cytosine within the recognition sequence is methylated at position C5 include, e.g., Aat II, Aci I, Acd I, Age I, Alu I, Asc I, Ase I, AsiS I, Bbe I, BsaA I, BsaH I, BsiE I, BsiW L BsrF I, BssH II, BssK I, BstB I, BstN I, BstU I, Cla I, Eae I, Eag I, Fau I, Fse I, Hha I, HinPl I, HinC II, Hpa II, Hpy99 I, HpyCH4 IV, Kas I, Mbo I, Mlu I, MapAl I, Msp, Nae I, Nar I, Not I, Pml I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfl I, Sfo I, SgrA I, Sma I, SnaB I, Tsc I, Xma I, and Zra I. Suitable methylation-sensitive restriction enzymes that do not cleave DNA at or near their recognition sequence when an adenosine within the recognition sequence is methylated at position N6 include, e.g., Mbo I. One of skill in the art will appreciate that any methylation-sensitive restriction enzyme, including homologs and orthologs of the restriction enzymes described herein, is also suitable for use with one or more of the methods described herein. One of skill in the art will further appreciate that a methylation-sensitive restriction enzyme that fails to cut in the presence of methylation of a cytosine at or near its recognition sequence may be insensitive to the presence of methylation of an adenosine at or near its recognition sequence. Likewise, a methylation-sensitive restriction enzyme that fails to cut in the presence of methylation of an adenosine at or near its recognition sequence may be insensitive to the presence of methylation of a cytosine at or near its recognition sequence. For example, Sau3AI is sensitive (i.e., fails to cut) to the presence of a methylated cytosine at or near its recognition sequence, but is insensitive (i.e., cuts) to the presence of a methylated adenosine at or near its recognition sequence. One of skill in the art will also appreciate that some methylation-sensitive restriction enzymes are blocked by methylation of bases on one or both strands of DNA encompassing of their recognition sequence, while other methylation-sensitive restriction enzymes are blocked only by methylation on both strands, but can cut if a recognition site is hemi-methylated.

In alternative embodiments, adaptors are optionally added to the ends of the randomly fragmented DNA, the DNA is then digested with a methylation-dependent or methylation-sensitive restriction enzyme, and intact DNA is subsequently amplified using primers that hybridize to the adaptor sequences. In this case, a second step is performed to determine the presence, absence or quantity of a particular gene in an amplified pool of DNA. In some embodiments, the DNA is amplified using real-time, quantitative PCR.

In other embodiments, the methods comprise quantifying the average methylation density in a target sequence within a population of genomic DNA. In some embodiments, the method comprises contacting genomic DNA with a methylation-dependent restriction enzyme or methylation-sensitive restriction enzyme under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved; quantifying intact copies of the locus; and comparing the quantity of amplified product to a control value representing the quantity of methylation of control DNA, thereby quantifying the average methylation density in the locus compared to the methylation density of the control DNA.

In some instances, the quantity of methylation of a locus of DNA is determined by providing a sample of genomic DNA comprising the locus, cleaving the DNA with a restriction enzyme that is either methylation-sensitive or methylation-dependent, and then quantifying the amount of intact DNA or quantifying the amount of cut DNA at the DNA locus of interest. The amount of intact or cut DNA will depend on the initial amount of genomic DNA containing the locus, the amount of methylation in the locus, and the number (i.e., the fraction) of nucleotides in the locus that are methylated in the genomic DNA. The amount of methylation in a DNA locus can be determined by comparing the quantity of intact DNA or cut DNA to a control value representing the quantity of intact DNA or cut DNA in a similarly-treated DNA sample. The control value can represent a known or predicted number of methylated nucleotides. Alternatively, the control value can represent the quantity of intact or cut DNA from the same locus in another (e.g., normal, non-diseased) cell or a second locus.

By using at least one methylation-sensitive or methylation-dependent restriction enzyme under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved and subsequently quantifying the remaining intact copies and comparing the quantity to a control, average methylation density of a locus can be determined. If the methylation-sensitive restriction enzyme is contacted to copies of a DNA locus under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved, then the remaining intact DNA will be directly proportional to the methylation density, and thus may be compared to a control to determine the relative methylation density of the locus in the sample. Similarly, if a methylation-dependent restriction enzyme is contacted to copies of a DNA locus under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved, then the remaining intact DNA will be inversely proportional to the methylation density, and thus may be compared to a control to determine the relative methylation density of the locus in the sample. Such assays are disclosed in, e.g., U.S. Pat. No. 7,910,296.

The methylated CpG island amplification (MCA) technique is a method that can be used to screen for altered methylation patterns in genomic DNA, and to isolate specific sequences associated with these changes (Toyota et al, 1999, Cancer Res. 59, 2307-2312, U.S. Pat. No. 7,700,324 (Issa et al)). Briefly, restriction enzymes with different sensitivities to cytosine methylation in their recognition sites are used to digest genomic DNAs from primary tumors, cell lines, and normal tissues prior to arbitrarily primed PCR amplification. Fragments that show differential methylation are cloned and sequenced after resolving the PCR products on high-resolution polyacrylamide gels. The cloned fragments are then used as probes for Southern analysis to confirm differential methylation of these regions. Typical reagents (e.g., as might be found in a typical MCA-based kit) for MCA analysis may include, but are not limited to: PCR primers for arbitrary priming Genomic DNA; PCR buffers and nucleotides, restriction enzymes and appropriate buffers; gene-hybridization oligos or probes; control hybridization oligos or probes.

Additional methylation detection methods include those methods described in, e.g., U.S. Pat. Nos. 7,553,627; 6,331,393; U.S. patent Ser. No. 12/476,981; U.S. Patent Publication No. 2005/0069879; Rein, et al, 26(10) NUCLEIC ACIDS RES. 2255-64 (1998); and Olek et al. 17(3) NAT. GENET. 275-6 (1997).

In another embodiment, the methylation status of selected CpG sites is determined using Methylation-Sensitive High Resolution Melting (HRM). Recently, Wojdacz et al. reported methylation-sensitive high resolution melting as a technique to assess methylation. (Wojdacz and Dobrovic, 2007, Nuc. Acids Res. 35(6) e41; Wojdacz et al. 2008, Nat. Prot. 3(12) 1903-1908; Balic et al, 2009 J. Mol. Diagn. 11 102-108; and US Pat. Pub. No. 2009/0155791 (Wojdacz et al)). A variety of commercially available real time PCR machines have HRM systems including the Roche LightCycler480, Corbett Research RotorGene6000, and the Applied Biosystems 7500. HRM may also be combined with other amplification techniques such as pyrosequencing as described by Candiloro et al. (Candiloro et al, 2011, Epigenetics 6(4) 500-507).

In another embodiment, the methylation status of selected CpG locus is determined using a primer extension assay, including an optimized PCR amplification reaction that produces amplified targets for analysis using mass spectrometry. The assay can also be done in multiplex. Mass spectrometry is a particularly effective method for the detection of polynucleotides associated with the differentially methylated regulatory elements. The presence of the polynucleotide sequence is verified by comparing the mass of the detected signal with the expected mass of the polynucleotide of interest. The relative signal strength, e.g., mass peak on a spectra, for a particular polynucleotide sequence indicates the relative population of a specific allele, thus enabling calculation of the allele ratio directly from the data. This method is described in detail in PCT Pub. No. WO 2005/012578A1 (Beaulieu et al). For methylation analysis, the assay can be adopted to detect bisulfite introduced methylation dependent C to T sequence changes. These methods are particularly useful for performing multiplexed amplification reactions and multiplexed primer extension reactions (e.g., multiplexed homogeneous primer mass extension (hME) assays) in a single well to further increase the throughput and reduce the cost per reaction for primer extension reactions.

Other methods for DNA methylation analysis include restriction landmark genomic scanning (RLGS, Costello et al, 2002, Meth. Mol Biol, 200, 53-70), methylation-sensitive-representational difference analysis (MS-RDA, Ushijima and Yamashita, 2009, Methods Mol Biol 507, 117-130). Comprehensive high-throughput arrays for relative methylation (CHARM) techniques are described in WO 2009/021141 (Feinberg and Irizarry). The Roche® NimbleGen® microarrays including the Chromatin Immunoprecipitation-on-chip (ChlP-chip) or methylated DNA immunoprecipitation-on-chip (MeDIP-chip). These tools have been used for a variety of cancer applications including melanoma, liver cancer and lung cancer (Koga et al. 2009, Genome Res., 19, 1462-1470; Acevedo et al, 2008, Cancer Res., 68, 2641-2651; Rauch et al, 2008, Proc. Nat. Acad. Sci. USA, 105, 252-257). Others have reported bisulfate conversion, padlock probe hybridization, circularization, amplification and next generation or multiplexed sequencing for high throughput detection of methylation (Deng et al, 2009, Nat. Biotechnol 27, 353-360; Ball et al, 2009, Nat. Biotechnol 27, 361-368; U.S. Pat. No. 7,611,869 (Fan)). As an alternative to bisulfate oxidation, Bayeyt et al. have reported selective oxidants that oxidize 5-methylcytosine, without reacting with thymidine, which are followed by PCR or pyro sequencing (WO 2009/049916 (Bayeyt et al).

In some instances, quantitative amplification methods (e.g., quantitative PCR or quantitative linear amplification) are used to quantify the amount of intact DNA within a locus flanked by amplification primers following restriction digestion. Methods of quantitative amplification are disclosed in. e.g., U.S. Pat. Nos. 6,180,349; 6,033,854; and 5,972,602, as well as in, e.g., DeGraves, et al, 34(1) BIOTECHNIQUES 106-15 (2003); Deiman B, et al., 20(2) MOL. BIOTECHNOL. 163-79 (2002); and Gibson t al, 6 GENOME RESEARCH 995-1001 (1996).

Following reaction or separation of nucleic acid in a methylation specific manner, the nucleic acid in some cases are subjected to sequence-based analysis. For example, once it is determined that one particular genomic sequence from an aged sample is hypermethylated or hypomethylated compared to its counterpart, the amount of this genomic sequence can be determined. Subsequently, this amount can be compared to a standard control value and used to determine the biological age of the sample. In many instances, it is desirable to amplify a nucleic acid sequence using any of several nucleic acid amplification procedures which are well known in the art. Specifically, nucleic acid amplification is the chemical or enzymatic synthesis of nucleic acid copies which contain a sequence that is complementary to a nucleic acid sequence being amplified (template). The methods and kits may use any nucleic acid amplification or detection methods known to one skilled in the art, such as those described in U.S. Pat. No. 5,525,462 (Takarada et al); U.S. Pat. No. 6,114,117 (Hepp et al); U.S. Pat. No. 6,127,120 (Graham et al); U.S. Pat. No. 6,344,317 (Umovitz); U.S. Pat. No. 6,448,001 (Oku); U.S. Pat. No. 6,528,632 (Catanzariti et al); and PCT Pub. No. WO 2005/111209 (Nakajima et al).

In some embodiments, the nucleic acids are amplified by PCR amplification using methodologies known to one skilled in the art. One skilled in the art will recognize, however, that amplification can be accomplished by any known method, such as ligase chain reaction (LCR), Q-replicas amplification, rolling circle amplification, transcription amplification, self-sustained sequence replication, nucleic acid sequence-based amplification (NASBA), each of which provides sufficient amplification. Branched-DNA technology is also optionally used to qualitatively demonstrate the presence of a sequence of the technology, which represents a particular methylation pattern, or to quantitatively determine the amount of this particular genomic sequence in a sample. Nolte reviews branched-DNA signal amplification for direct quantitation of nucleic acid sequences in clinical samples (Nolte, 1998, Adv. Clin. Chem. 33:201-235).

The PCR process is well known in the art and include, for example, reverse transcription PCR, ligation mediated PCR, digital PCR (dPCR), or droplet digital PCR (ddPCR). For a review of PCR methods and protocols, see, e.g., Innis et al, eds., PCR Protocols, A Guide to Methods and Application, Academic Press, Inc., San Diego, Calif. 1990; U.S. Pat. No. 4,683,202 (Mullis). PCR reagents and protocols are also available from commercial vendors, such as Roche Molecular Systems. In some instances, PCR is carried out as an automated process with a thermostable enzyme. In this process, the temperature of the reaction mixture is cycled through a denaturing region, a primer annealing region, and an extension reaction region automatically. Machines specifically adapted for this purpose are commercially available.

In some embodiments, amplified sequences are also measured using invasive cleavage reactions such as the Invader® technology (Zou et al, 2010, Association of Clinical Chemistry (AACC) poster presentation on Jul. 28, 2010, “Sensitive Quantification of Methylated Markers with a Novel Methylation Specific Technology; and U.S. Pat. No. 7,011,944 (Prudent et al)).

Suitable next generation sequencing technologies are widely available. Examples include the 454 Life Sciences platform (Roche, Branford, Conn.) (Margulies et al. 2005 Nature, 437, 376-380); Illumina's Genome Analyzer, GoldenGate Methylation Assay, or Infinium Methylation Assays, i.e., Infinium HumanMethylation 27K BeadArray or VeraCode GoldenGate methylation array (Illumina, San Diego, Calif.; Bibkova et al, 2006, Genome Res. 16, 383-393; U.S. Pat. Nos. 6,306,597 and 7,598,035 (Macevicz); U.S. Pat. No. 7,232,656 (Balasubramanian et al.)); QX200™ Droplet Digital™ PCR System from Bio-Rad; or DNA Sequencing by Ligation, SOLiD System (Applied Biosystems/Life Technologies; U.S. Pat. Nos. 6,797,470, 7,083,917, 7,166,434, 7,320,865, 7,332,285, 7,364,858, and 7,429,453 (Barany et al); the Helicos True Single Molecule DNA sequencing technology (Harris et al, 2008 Science, 320, 106-109; U.S. Pat. Nos. 7,037,687 and 7,645,596 (Williams et al); U.S. Pat. No. 7,169,560 (Lapidus et al); U.S. Pat. No. 7,769,400 (Harris)), the single molecule, real-time (SMRT™) technology of Pacific Biosciences, and sequencing (Soni and Meller, 2007, Clin. Chem. 53, 1996-2001); semiconductor sequencing (Ion Torrent; Personal Genome Machine); DNA nanoball sequencing; sequencing using technology from Dover Systems (Polonator), and technologies that do not require amplification or otherwise transform native DNA prior to sequencing (e.g., Pacific Biosciences and Helicos), such as nanopore-based strategies (e.g., Oxford Nanopore, Genia Technologies, and Nabsys). These systems allow the sequencing of many nucleic acid molecules isolated from a specimen at high orders of multiplexing in a parallel fashion. Each of these platforms allow sequencing of clonally expanded or non-amplified single molecules of nucleic acid fragments. Certain platforms involve, for example, (i) sequencing by ligation of dye-modified probes (including cyclic ligation and cleavage), (ii) pyrosequencing, and (iii) single-molecule sequencing.

Pyrosequencing is a nucleic acid sequencing method based on sequencing by synthesis, which relies on detection of a pyrophosphate released on nucleotide incorporation. Generally, sequencing by synthesis involves synthesizing, one nucleotide at a time, a DNA strand complimentary to the strand whose sequence is being sought. Study nucleic acids may be immobilized to a solid support, hybridized with a sequencing primer, incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5′ phosphsulfate and luciferin. Nucleotide solutions are sequentially added and removed. Correct incorporation of a nucleotide releases a pyrophosphate, which interacts with ATP sulfurylase and produces ATP in the presence of adenosine 5′ phosphsulfate, fueling the luciferin reaction, which produces a chemiluminescent signal allowing sequence determination. Machines for pyrosequencing and methylation specific reagents are available from Qiagen, Inc. (Valencia, Calif.). See also Tost and Gut, 2007, Nat. Prot. 2 2265-2275. An example of a system that can be used by a person of ordinary skill based on pyrosequencing generally involves the following steps: ligating an adaptor nucleic acid to a study nucleic acid and hybridizing the study nucleic acid to a bead; amplifying a nucleotide sequence in the study nucleic acid in an emulsion; sorting beads using a picoliter multiwell solid support, and sequencing amplified nucleotide sequences by pyrosequencing methodology (e.g., Nakano et al, 2003, J. Biotech. 102, 117-124). Such a system can be used to exponentially amplify amplification products generated by a process described herein, e.g., by ligating a heterologous nucleic acid to the first amplification product generated by a process described herein.

CpG Methylation Data Analysis Methods

In certain embodiments, the methylation values measured for markers of an epigenetic marker panel are mathematically combined and the combined value is correlated to the underlying diagnostic question. In some instances, methylated marker values are combined by any appropriate state of the art mathematical method. Well-known mathematical methods for correlating a marker combination to a disease status employ methods like discriminant analysis (DA) (e.g., linear-, quadratic-, regularized-DA), Discriminant Functional Analysis (DFA), Kernel Methods (e.g., SVM), Multidimensional Scaling (MDS), Nonparametric Methods (e.g., k-Nearest-Neighbor Classifiers), PLS (Partial Least Squares), Tree-Based Methods (e.g., Logic Regression, CART, Random Forest Methods, Boosting/Bagging Methods), Generalized Linear Models (e.g., Logistic Regression), Principal Components based Methods (e.g., SIMCA), Generalized Additive Models, Fuzzy Logic based Methods, Neural Networks and Genetic Algorithms based Methods. The skilled artisan will have no problem in selecting an appropriate method to evaluate an epigenetic marker or marker combination described herein. In one embodiment, the method used in a correlating methylation status of an epigenetic marker or marker combination, e.g. to diagnose a cancer or an aging-related disease or disorder, is selected from DA (e.g., Linear-, Quadratic-, Regularized Discriminant Analysis), DFA, Kernel Methods (e.g., SVM), MDS, Nonparametric Methods (e.g., k-Nearest-Neighbor Classifiers), PLS (Partial Least Squares), Tree-Based Methods (e.g., Logic Regression, CART, Random Forest Methods, Boosting Methods), or Generalized Linear Models (e.g., Logistic Regression), and Principal Components Analysis. Details relating to these statistical methods are found in the following references: Ruczinski et al., 12 J. OF COMPUTATIONAL AND GRAPHICAL STATISTICS 475-511 (2003); Friedman, J. H., 84 J. OF THE AMERICAN STATISTICAL ASSOCIATION 165-75 (1989); Hastie, Trevor, Tibshirani, Robert, Friedman, Jerome, The Elements of Statistical Learning, Springer Series in Statistics (2001); Breiman, L., Friedman, J. H., Olshen. R. A., Stone, C. J. Classification and regression trees, California: Wadsworth (1984); Breiman, L., 45 MACHINE LEARNING 5-32 (2001); Pepe, M. S., The Statistical Evaluation of Medical Tests for Classification and Prediction, Oxford Statistical Science Series, 28 (2003); and Duda, R. O., Hart. P. E., Stork, D. O., Pattern Classification, Wiley Interscience, 2nd Edition (2001).

In one embodiment, the correlated results for each methylation panel are rated by their correlation to the disease or tumor type positive state, such as for example, by p-value test or t-value test or F-test. Rated (best first, i.e. low p- or t-value) markers are then subsequently selected and added to the methylation panel until a certain diagnostic value is reached. Such methods include identification of methylation panels, or more broadly, genes that were differentially methylated among several classes using, for example, a random-variance t-test (Wright G. W. and Simon R, Bioinformatics 19:2448-2455, 2003). Other methods include the step of specifying a significance level to be used for determining the epigenetic markers that will be included in the marker panel. Epigenetic markers that are differentially methylated between the classes at a univariate parametric significance level less than the specified threshold are included in the panel. It doesn't matter whether the specified significance level is small enough to exclude enough false discoveries. In some problems better prediction is achieved by being more liberal about the marker panels used as features. In some cases, the panels are biologically interpretable and clinically applicable, however, if fewer markers are included. Similar to cross-validation, marker selection is repeated for each training set created in the cross-validation process. That is for the purpose of providing an unbiased estimate of prediction error. The methylation panel for use with new patient sample data is the one resulting from application of the methylation selection and classifier of the “known” methylation information, or control methylation panel.

In some embodiments, models for utilizing methylation profile to predict the class of future samples are also used. In some cases, these models are based on the Compound Covariate Predictor (Radmacher et al. Journal of Computational Biology 9:505-511, 2002), Diagonal Linear Discriminant Analysis (Dudoit et al. Journal of the American Statistical Association 97:77-87, 2002), Nearest Neighbor Classification (also Dudoit et al.), and Support Vector Machines with linear kernel (Ramaswamy et al. PNAS USA 98:15149-54, 2001). The models incorporated markers that were differentially methylated at a given significance level (e.g. 0.01, 0.05 or 0.1) as assessed by the random variance t-test (Wright G. W. and Simon R. Bioinformatics 19:2448-2455, 2003). The prediction error of each model using cross validation, preferably leave-one-out cross-validation (Simon et al. Journal of the National Cancer Institute 95:14-18, 2003 is optinally estimated. For each leave-one-out cross-validation training set, the entire model building process is repeated, including the epigenetic marker selection process. It may also be evaluated whether the cross-validated error rate estimate for a model is significantly less than expected from random prediction. The class labels can be randomly permuted and the entire leave-one-out cross-validation process is then repeated. The significance level is the proportion of the random permutations that gives a cross-validated error rate no greater than the cross-validated error rate obtained with the real methylation data.

Another classification method is the greedy-pairs method described by Bo and Jonassen (Genome Biology 3(4):research0017.1-0017.11, 2002). The greedy-pairs approach starts with ranking all markers based on their individual t-scores on the training set. This method attempts to select pairs of markers that work well together to discriminate the classes.

Furthermore, a binary tree classifier for utilizing methylation profile can be used to predict the class of future samples. The first node of the tree incorporated a binary classifier that distinguished two subsets of the total set of classes. The individual binary classifiers are based on the “Support Vector Machines” incorporating markers that were differentially expressed among markers at the significance level (e.g. 0.01, 0.05 or 0.1) as assessed by the random variance t-test (Wright G. W. and Simon R. Bioinformatics 19:2448-2455, 2003). Classifiers for all possible binary partitions are evaluated and the partition selected is that for which the cross-validated prediction error is minimum. The process is then repeated successively for the two subsets of classes determined by the previous binary split. The prediction error of the binary tree classifier can be estimated by cross-validating the entire tree building process. This overall cross-validation includes re-selection of the optimal partitions at each node and re-selection of the markers used for each cross-validated training set as described by Simon et al. (Simon et al. Journal of the National Cancer Institute 95:14-18, 2003). Several-fold cross validation in which a fraction of the samples is withheld, a binary tree developed on the remaining samples, and then class membership is predicted for the samples withheld. This is repeated several times, each time withholding a different percentage of the samples. The samples are randomly partitioned into fractional test sets (Simon R and Lam A. BRB-ArrayTools User Guide, version 3.2. Biometric Research Branch, National Cancer Institute).

Thus, in one embodiment, the correlated results for each marker b) are rated by their correct correlation to the disease, preferably by p-value test. It is also possible to include a step in that the markers are selected d) in order of their rating.

In additional embodiments, factors such as the value, level, feature, characteristic, property, etc. of a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be utilized in addition prior to, during, or after administering a therapy to a patient to enable further analysis of the patient's cancer status.

In some embodiments, a diagnostic test to correctly predict status is measured as the sensitivity of the assay, the specificity of the assay or the area under a receiver operated characteristic (“ROC”) curve. In some instances, sensitivity is the percentage of true positives that are predicted by a test to be positive, while specificity is the percentage of true negatives that are predicted by a test to be negative. In some cases, an ROC curve provides the sensitivity of a test as a function of 1-specificity. The greater the area under the ROC curve, for example, the more powerful the predictive value of the test. Other useful measures of the utility of a test include positive predictive value and negative predictive value. Positive predictive value is the percentage of people who test positive that are actually positive. Negative predictive value is the percentage of people who test negative that are actually negative.

In some embodiments, one or more of the epigenetic biomarkers disclosed herein show a statistical difference in different samples of at least p<0.05, p<10⁻², p<10⁻³, p<10⁻⁴, or p<10⁻⁵. Diagnostic tests that use these biomarkers may show an ROC of at least 0.6, at least about 0.7, at least about 0.8, or at least about 0.9. The biomarkers are differentially methylated in different subjects with different ages, and the biomarkers for each age range are differentially methylated, and, therefore, are useful in aiding in the determination of a subject's biological age (or bioage) and its correlation to chronological age. In certain embodiments, the biomarkers are measured in a patient sample using the methods described herein and compared, for example, to predefined biomarker levels and correlated to the patient's chronological age. In other embodiments, the correlation of a combination of biomarkers in a patient sample is compared, for example, to a predefined set ofbiomarkers. In some embodiments, the measurement(s) is then compared with a relevant diagnostic amount(s), cut-off(s), or multivariate model scores that distinguish between different biological ages. As is well understood in the art, by adjusting the particular diagnostic cut-off(s) used in an assay, one can increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician. In some embodiments, the particular diagnostic cut-off is determined, for example, by measuring the amount of biomarker hypermethylation or hypomethylation in a statistically significant number of samples from patients with different ages, and drawing the cut-off to suit the desired levels of specificity and sensitivity.

Kits/Article of Manufacture

In some embodiments, provided herein include kits for detecting and/or characterizing the expression level and/or methylation profile of an epigenetic marker described herein. In some instances, the kit comprises a plurality of primers or probes to detect or measure the methylation status/levels of one or more samples. Such kits comprise, in some instances, at least one polynucleotide that hybridizes to at least one of the methylation marker sequences described herein and at least one reagent for detection of gene methylation. Reagents for detection of methylation include, e.g., sodium bisulfate, polynucleotides designed to hybridize to sequence that is the product of a marker sequence if the marker sequence is not methylated (e.g., containing at least one C-U conversion), and/or a methylation-sensitive or methylation-dependent restriction enzyme. In some cases, the kits provide solid supports in the form of an assay apparatus that is adapted to use in the assay. In some instances, the kits further comprise detectable labels, optionally linked to a polynucleotide, e.g., a probe, in the kit.

In some embodiments, the kits comprise one or more (e.g., 1, 2, 3, 4, or more) different polynucleotides (e.g., primers and/or probes) capable of specifically amplifying at least a portion of a DNA region of an epigenetic marker described herein. Optionally, one or more detectably-labeled polypeptides capable of hybridizing to the amplified portion are also included in the kit. In some embodiments, the kits comprise sufficient primers to amplify 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different DNA regions or portions thereof, and optionally include detectably-labeled polynucleotides capable of hybridizing to each amplified DNA region or portion thereof. The kits further can comprise a methylation-dependent or methylation sensitive restriction enzyme and/or sodium bisulfite.

In some embodiments, the kits comprise sodium bisulfite, primers and adapters (e.g., oligonucleotides that can be ligated or otherwise linked to genomic fragments) for whole genome amplification, and polynucleotides (e.g., detectably-labeled polynucleotides) to quantify the presence of the converted methylated and or the converted unmethylated sequence of at least one cytosine from a DNA region of an epigenetic marker described herein.

In some embodiments, the kits comprise methylation sensing restriction enzymes (e.g., a methylation-dependent restriction enzyme and/or a methylation-sensitive restriction enzyme), primers and adapters for whole genome amplification, and polynucleotides to quantify the number of copies of at least a portion of a DNA region of an epigenetic marker described herein.

In some embodiments, the kits comprise a methylation binding moiety and one or more polynucleotides to quantify the number of copies of at least a portion of a DNA region of a marker described herein. A methylation binding moiety refers to a molecule (e.g., a polypeptide) that specifically binds to methyl-cytosine.

Examples include restriction enzymes or fragments thereof that lack DNA cutting activity but retain the ability to bind methylated DNA, antibodies that specifically bind to methylated DNA, etc.).

In some embodiments, the kit includes a packaging material. As used herein, the term “packaging material” can refer to a physical structure housing the components of the kit. In some instances, the packaging material maintains sterility of the kit components, and is made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, etc.). Other materials useful in the performance of the assays are included in the kits, including test tubes, transfer pipettes, and the like. In some cases, the kits also include written instructions for the use of one or more of these reagents in any of the assays described herein.

In some embodiments, kits also include a buffering agent, a preservative, or a protein/nucleic acid stabilizing agent. In some cases, kits also include other components of a reaction mixture as described herein. For example, kits include one or more aliquots of thermostable DNA polymerase as described herein, and/or one or more aliquots of dNTPs. In some cases, kits also include control samples of known amounts of template DNA molecules harboring the individual alleles of a locus. In some embodiments, the kit includes a negative control sample, e.g., a sample that does not contain DNA molecules harboring the individual alleles of a locus. In some embodiments, the kit includes a positive control sample, e.g., a sample containing known amounts of one or more of the individual alleles of a locus.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the general description and the detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term “about” includes an amount that would be expected to be within experimental error.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly or a hospice worker).

A “site” corresponds to a single site, which in some cases is a single base position or a group of correlated base positions, e.g., a CpG site. A “locus” corresponds to a region that includes multiple sites. In some instances, a locus includes one site.

As used herein, the terms “biological age (bioage),” “chemical age,” “methylomic age,” and “molecular age” are equivalent or synonymous. The biological age is determined using a set of age-associated markers (e.g., epigenetic markers) of a subject or an organism. In the current disclosure, the biological age is determined from an analysis of the modification status of specific CpG dinucleotide and, in particular, e.g., the methylation status at the C-5 position of cytosine.

Chronological age is the actual age of a subject or organism. In some instances, for animals and humans, chronological age is based on the age calculated from the moment of conception or based on the age calculated from the time and date of birth. The chronological age of the cell, tissue or organ may be determined from the chronological age of the subject or organism from which the cell, tissue or organ is obtained, plus the duration of the cell, tissue or organ is placed in culture. Alternatively, in the case of the cell or tissue culture, the chronological age may be related to the total or accumulative time in culture or passage number.

EXAMPLES

These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1. Effect of Vitamin C on Senescence and Proliferation in Human Fibroblasts

The proliferation effects of vitamin C on WI38 fibroblasts were tested. This cell line is useful as an aging model since it is a mortal human cell line that follows the Hayflick limit, in that it undergoes a certain number of cell divisions before stopping. The senescence level in WI38 increased as the cell line divided. Vitamin C and its derivative were tested to determine whether they would increase the proliferation rate of fibroblast cells and upregulate an established age-related marker (ARM), e.g., ELOVL2, which was found to decrease with age in WI38 fibroblasts. A membrane-soluble derivative of vitamin C, 6-O-Palmitoyl-L-ascorbic acid (PalmAA), which has an additional fatty acid that allows it to pass through the cell membrane, was also tested. The oxidized derivative of vitamin C, dehydroascorbic acid (DHAA), as Vitamin C is actively converted to DHAA in cell culture media, was tested as well. Insulin was also added to this experiment to upregulate Glut-1 transporter, through which DHAA enters the cell.

Vitamin C induced a dose-dependent response on WI38 fibroblasts. Low concentrations of vitamin C (50 μg/mL to 100 μg/mL) induced fibroblast proliferation while higher concentrations of vitamin C (200 μg/mL to 500 μg/mL) slowed or inhibited fibroblast cell growth while causing cell death. Furthermore, the proliferative effect of 50 μg/mL vitamin C was more pronounced on older fibroblast cells compared to younger fibroblast cells. 6-O-Palmitoyl L-ascorbic acid, a derivative of vitamin C that is permeable to the cell membrane, induced minimal proliferation and caused no gene expression change in the age related marker. On the other hand, dehydroascorbic acid (DHAA), the oxidized form of vitamin C, induced lower cell proliferation compared to an equal concentration of vitamin C.

FIG. 1 shows phenotypic and genotypic effects of concentration dependent vitamin C treatment were analyzed on WI38 PD46 and 48 fibroblast cells. A) Cell images of 12-well plate treated with low concentration vitamin C at Day 0, 4 and 5 for PD46. B) Confluency plot calculated through ImageJ of PD46, n=2. C) and D) Expression graphs for ARM (e.g., ELOVL2) and SLC2A1 for PD46, n=3. E) Cell images of 12-well treated with high concentration vitamin C at Day 0, 4 and 5 for PD48. F) Confluency plot calculated through ImageJ of PD48, n=2. G) and I) Expression graphs for ARM and SLC2A1 for PD48, n=3.

FIG. 2 illustrates phenotypic and genotypic effects of vitamin C treatment were analyzed on younger WI38 PD42 and older WI38 PD58 fibroblasts. A) Cell images of 12-well at Day 0, 1 and 2 of treatment for PD42. B) Confluency plot calculated through ImageJ of PD42, n=2. C) and D) Expression graphs for ARM (e.g., ELOVL2) and SLC2A1 for PD42, n=3. E) Cell images of 12-well at Day 0, 5 and 7 of treatment for PD58. F) Confluency plot calculated through ImageJ of PD58, n=2. G) and H) Expression graphs for ARM (e.g., ELOVL2) and SLC2A1 for PD53, n=3. I) Cell images of senescence and DAPI staining of PD45.5 fibroblasts. J) Graph of percentage senescence for younger PD32 fibroblast and older PD45.5 fibroblast. n=3.

FIG. 3 shows phenotypic and genotypic effects of 6-O-Palmitoyl L-ascorbic acid treatment were analyzed on younger WI38 PD55 fibroblasts. A) Cell images of 12-well at Day 0 and Day 8 of treatment for PD55. B) Confluency plot calculated through ImageJ for PD55, n=2. C) and D) Expression graph for ARM (e.g., ELOVL2) and SLC2A1 for PD55, n=3

FIG. 4 shows phenotypic and genotypic effects of dehydroascorbic acid and vitamin C treatment complemented with the addition of insulin were analyzed on WI38 PD54 fibroblast cells. A) Diagram of postulated pathway for interconversion of DHAA to vitamin C and their effect on fibroblast cells. B) Cell images of 12-well at Day 10 of treatment for PD54. C) Confluency plot calculated through ImageJ of PD54, n=2. D and E) Expression graphs for ARM (e.g., ELOVL2) and SLC2A1 for PD54, n=3. F) Graph of percentage senescence for younger PD32 fibroblast and older PD45.5 fibroblast. n=3. G) Fluorescent ROS assay showing fluorescent ROS relative to total fibroblasts in PD48 fibroblasts.

Example 2. Diabetes and Progeria Affect Biological Aging Rate

In some embodiments, it was shown that patients with diabetes or progeria have an accelerated biological aging rate.

FIG. 5 illustrates that patients with diabetes have an older biological age than patients who do not have diabetes. In some instances, patients with type I diabetes (T1DM) are about 12% older in biological age than normal patients. In some cases, patients with type II diabetes (T2DM) are about 5% older in biological age than normal patients.

FIG. 6A illustrates the correlation of BMI with biological age. In some cases, as the BMI increases, the rate of increase in biological aging also increases.

FIG. 6B illustrates the correlation of biological aging between male and female. In some cases, male is about 1% older in biological age than female.

FIG. 7 shows biological age prediction using an exemplar) 71 methylation markers in three progeria cell lines. Each biological age (bioage) is higher than chronological age.

Example 3. Environmental Factors Affect Biological Aging Rate

In some embodiments, it was shown that external influences such as environmental factors further modulates the biological aging rate.

FIG. 8 shows that external influences, such as diet and exercise, in some cases, reverses biological age in a 6 month trial. Additional external influences such as stress and/or pharmacologies further influences biological aging.

FIG. 9 shows an exemplary list of genes and CpG sites that are utilized for biological age prediction.

Example 4. Methylation Level and Expression Level of Epigenetic Markers ELOVL2 and KLF14 Changes with Age

FIG. 10 shows a decrease in expression of ELOVL2 and KLF14 in older fibroblasts.

FIG. 11 shows a decrease in expression of ELOVL2 in cell line IMR90 (A) and cell line WI38 (B).

FIG. 12 shows the expression level of ELOVL2 and KLF14 in human blood (A), a human fibroblast cell line WI38 (B) and human lens tissue (C).

FIG. 13 shows the expression level of an exemplary list of genes.

FIG. 14A-FIG. 14C shows the biological age (or methylation age) increases with age. FIG. 14A shows the biological age increases with cell line population doubling. FIG. 14B shows the increase in methylation level of ELOVL2, PENK, and KLF14. FIG. 14C shows the increase in methylation level of FHL2 and SMC4.

FIG. 15 shows human KLF14 locus showing methylation CpG islands.

FIG. 16 shows human ELOVL2 locus showing methylation CpG islands.

Example 5. Knockdown of KLF14 and ELOVL2 Increases Cellular Aging and Senescence and Reduces Cell Proliferation

FIG. 17 shows ELOVL2 knockdown efficiency in three cell lines: WI38 (A). IMR90 (B), and 293T (C).

FIG. 18A-FIG. 18D show that ELOVL2 knockdown reduces cell proliferation. FIG. 18A shows a decrease of cells in ELOVL2 knockdown relative to the control (shLuc) in all three cell lines, WI38, IMR90, and 293T. FIG. 18B-FIG. 18D show the PD45 confluency of ELOVL2 knockdown relative to the control (shLuc) in the respective cell lines; WI38 (FIG. 18B), IMR90 (FIG. 18C), and 293T (FIG. 18D).

FIG. 19A-FIG. 19C show ELOVL2 knockdown increases senescence.

FIG. 20 shows ELOVL2 overexpression increases survival in old cells (PD56).

FIG. 21 shows knockdown of KLF14 in WI38 cells.

FIG. 22 shows the effect of KLF14 knockdown on other genes. The KLF14 knockdown is about 99.5%.

FIG. 23 illustrates the morphology of knockdown of ELOVL2 and KLF14 in cells.

FIG. 24 shows a senescence assay of the knockdown cells. As shown by a beta-gal assay, an increase in blue cells indicates that knockdown of ELOVL2 or KLF14 increases cell senescence.

Example 6. Incubation with Vitamin C Reduces Biological Age of Fibroblast and Reprograms Fibroblast into iPSC

WI38 cells at PD55 (55^(th) population doubling) were incubated with different concentrations of vitamin C (Vc), L-dehydro ascorbic acid (DHAA or DHA), or L-ascorbic acid 2-phosphate (VcP). DHAA (or DHA) is an oxidized form of vitamin C. L-ascorbic acid 2-phosphate (VcP) is a vitamin C derivative. Three concentrations were used for each tested compound and the concentrations included 0.3 mM (equivalent to 50 mG), 1.2 mM, or 1.8 mM.

A low concentration of vitamin C (at 0.3 mM) is observed to increase cell proliferation while a higher concentration of vitamin C (at 1.2 mM or 1.8 mM) is observed to have a slower cell proliferation rate relative to the concentration at 0.3 mM (FIG. 25A). An increased cell proliferation is not observed for DHAA (FIG. 25B). At all three concentrations of L-ascorbic acid 2-phosphate (VcP), cell proliferation is observed (FIG. 25C).

Similarly, a low concentration of vitamin C and all concentrations of L-ascorbic acid 2-phosphate (VcP) are observed as protective against cell senescence (FIG. 26) as measured by a betal gal staining assay. DHAA did not exert a protective effect against cell senescence.

The expression of ELOVL2 is also observed to be increased with a low concentration of vitamin C and all concentrations of L-ascorbic acid 2-phosphate (VcP) but not with DHAA (FIG. 27).

The biological age is also observed to be reversed in the presence of a low concentration of vitamin C and is reverted into iPSCs from aged fibroblast (FIG. 28).

Example 7. ELOVL2 and KLF14 Expression and Methylation Levels in a Mouse Model

ELOVL2 expression level was measured in different aged mouse tissue samples: liver, brain, lung and fatty tissue. ELOVL2 is observed to decrease with age (FIG. 29). Similarly in different aged mouse liver samples, ELOVL2 expression is observed as highest in young mice (age 12 days to 1 month) and lowest in old mice (age 1-2.3 years) (FIG. 30).

Expression of ELOVL2 in fibroblast cells of heterozygous knockout mice (8 bp frameshift, truncation) is decreased by about 50% and cell senescence (e.g., B-Gal positive) is increased by about 50% (FIG. 31).

In addition, the methylation level of ELOVL2 and KLF14 are measured in both young (12 days old) mice and old (2.3 years old) mice. The methylation level is observed to increase over age (FIG. 32).

Liver cell senescence in 2-yr. old Elovl2 heterozygous knockout mice (Het 83-2, Het-77-1, and Het 83-2) have much increased cell senescence compared to same age control mice (WT81-5, WT81-7) (FIG. 33A and FIG. 33B).

Het 83-2 Elovl2 heterozygous mouse exhibited dramatic aging phenotypes including hair loss, obesity, tumor formation (FIG. 34).

Example 8. Effect of ELOVL2 on Memory and Senescence

FIG. 37 shows that senescence and Elovl2 deletion affect the spatial memory of mice in a Morris water maze. After six days of training, the old wild type (WT-O, n=20) and the young Elovl2^(−/−) (−/− Y, n=20) mice show similar latency to reach the platforms while the young wild type (WT-Y, n=20) and the Elovl2^(+/−) (+/− Y, n=20) mice were all able to reach the platforms in a significantly shorter time. See FIG. 37A. In the first quadrant, 10/20 WT-O and 17/20 −/− Y mice failed to reach the platforms (WT-Y: 13.91±7.91 s; WT-O: 75.06±20.49 s; +/− Y: 35.78±21.79 s; −/− Y: 87.52±6.62 s). In the second quadrant, 10/20 WT-O and 11/20 −/− Y mice failed to reach the platforms (WT-Y: 12.38±5.32 s; WT-O: 62.93±30.46 s; +/− Y: 18.18±13.7 s; −/− Y: 76.62±19.09 s). In the third quadrant, 6/20 WT-O and 13/20 −/− Y mice failed to reach the platforms (WT-Y: 15.3±8.92 s; WT-O: 58.85±26.84 s; +/− Y: 16.53±10.41 s; −/− Y: 79.00±19.34 s). In the fourth quadrant, 6/20 WT-O and 8/20 −/− Y mice failed to reach the platforms (WT-Y: 4.74±3.25 s. WT-O: 45.24±38.66 s; +/− Y: 9.83±9.55 s; −/− Y: 63.54±29.50 s). The results demonstrated a poor spatial memory of the WT-O and the −/− Y mice. In FIG. 37B, the escape-platforms were removed at Day 7. The frequency of appearance for mice in the original locations of platforms was measured in 90 s. The WT-Y (n=20) and the +/− Y (n=20) mice have a higher frequency of appearance compared to the WT-O (n=20) and the −/− Y (n=20) mice. In the first quadrant, WT-Y: 7.50±1.57 times; WT-O: 1.30±1.34 times; +/− Y: 6.75±1.62 times; −/− Y: 0.95±1.43 times. In the second quadrant, WT-Y: 7.90±1.89 times; WT-O: 1.60±1.50 times; +/− Y: 6.55±2.42 times; −/− Y: 0.50±0.89 times. In the third quadrant, WT-Y: 7.10±2.17 times; WT-O: 1.45±1.10 times; +/− Y: 6.65±1.63 times; −/− Y: 0.55±0.89 times. In the fourth quadrant, WT-Y: 7.00±1.97 times; WT-O: 1.65±1.18 times; +/− Y: 6.35±1.98 times; −/− Y: 0.60±0.75 times. These data indicated the WT-O and the −/− Y mice have decreased long-term spatial reference memory. In FIG. 37C, the escape-platforms have been removed for two days at Day 8. All groups have shown diminished frequency of appearance in the original locations of platforms. The WT-Y (n=20) and the +/− Y (n=20) mice still have a higher frequency of appearance compared to the WT-O (n=20) and the −/− Y (n=20) mice. In the first quadrant, WT-Y: 3.60±0.88 times; WT-O: 0.40±0.60 times; +/− Y: 3.40±0.88 times; −/− Y: 0.30±0.57 times. In the second quadrant, WT-Y: 3.40±0.94 times; WT-O: 0.45±0.76 times; +/− Y: 3.40±1.27 times; −/− Y: 0.35±0.67 times. In the third quadrant, WT-Y: 3.10±0.85 times; WT-O: 0.70±0.80 times; +/− Y: 2.80±0.77 times; −/− Y: 0.50±0.76 times. In the fourth quadrant, WT-Y: 3.10±1.02 times; WT-O: 0.45±0.69 times; +/− Y: 3.5±0.95 times; −/− Y: 0.45±0.60 times. It further confirmed the reduction on long-term spatial reference memory of the WT-O and the −/− Y mice. Four difference locations of the platforms were tested in all the experiments.

FIG. 38 shows NAA/Cr and MI/Cr ratio, ADC, and Blood-perfusion (B-per) MRI analysis of wild type young (WT-Y) mice, wild type old (WT-O) mice, Elovl2 single (+/− Y) knock-out mice and Elovl2 double (−/− Y) knock-out mice. In hippocampus (FIG. 38A) and cortex (FIG. 38B), the ratio of NAA/Cr (N-acetylaspartate/Creatine) and MI/Cr both decrease a lot in WT-old and Elovl2 −/− mice show an increase of aged neuo-degenerative and loss of neuronal and Glial cells relative to WT-Y and Elovl2 +/− mice, indicating an increase of accelerated aging neuodegenerative phenotype and Alzheimer's Disease. ADC (apparent diffusion coefficient) shows that the diffusion of water molecules within tissue in WT-O, +/− Y and −/− Y mice has increased. B-per value shows that the Elovl2 −/− mice has the lowest blood flow relative to the other three groups of mice. NAA: neuronal cell marker, Cr: Energy metabolism, MI: Glial cell marker.

Example 9

Table 1A and Table 1B illustrate exemplary list of epigenetic markers for use with one or more methods described herein.

TABLE 1A UCSC_Ref SEQ Chro- Coor- Gene MAP ID mosome_ dinate_ Marker Coeff PTest Name CHR INFO SourceSeq NO: 36 36 cg16867657 170.5444 0 ELOVL2 6 11044877 CGGCGGCTCAACGT 1 6 11152863 CCACGGAGCCCCAG GAATACCCACCCGC TGCCCAGA cg10501210 -120.624 1.43E-294 1 207997020 CGGGACTGCGGCAC 2 1 206063643 CTTACGGCGGGACC AAGATTTGGGTCTG CGCAGGCG cg22454769 153.6159 2.92E-264 FHL2 2 106015767 CTTGGGAGCACAGT 3 2 105382199 AGTTATCGGGAGCG TCGCCTCCGGCGTG GGCTCTCG cg04875128 144.0318 1.87E-256 OTUD7A 15 31775895 CGCCACGTACCCGC 4 15 29563187 AGCAGAACCGCTCG CTGTCGTCGCAGAG CTACAGCC cg24724428 170.6561 5.72E-248 ELOVL2 6 11044888 CGTCCACGGAGCCC 5 6 11152874 CAGGAATACCCACC CGCTGCCCAGATCG GCAGCCGC cg06639320 192.5145 1.36E-223 FHL2 2 106015739 AGGGCTCCTTTCTT 6 2 105382171 CGTGCCCTCCGGGT CTTGGGAGCACAGT AGTTATCG cg14556683 215.314 1.20E-222 EPHX3 19 15342982 GAGAACACCAGGCT 7 19 15203982 CCACATGAAGGCGC GCAGCAGCTTCAGC GACAGGCG cg23606718 271.9213 7.41E-221 FAM123C 2 131513927 TCTCGGGGCCTTGG 8 2 131230397 CGACTTACCGCTGG GGGCCCGCAGTGCA GCAGGGCG cg07553761 222.4944 1.44E-217 TRIM59 3 160167977 CGCCGGTGGCCGAC 9 3 161650671 GGCTTCTGAGGAAT TATCTTTTACTTGG CGCCACAC cg14361627 259.4609 1.60E-214 KLF14 7 130419116 GCCCCCCGGCTAAG 10 7 130069656 TCATGTTTAACAGC CTCAGAAATTATCT TGTCTCCG cg14692377 298.8267 1.36E-213 SLC6A4 17 28562685 GGCTGCGCGGGGAG 11 17 25586811 GCTGGTCCCGGGCT GGGCAGGCGGGCTG GCCTCGCG cg19283806 -179.174 1.60E-213 CCDC102B 18 66389420 GATTTCTCCTTGAA 12 18 64540400 CAATCCCCGCAAAG ATAGCAGCCAAAAA AGGATGCG cg00292135 281.1003 3.60E-213 C7orf13; 7 156433068 AGGCCCAGGTGGGC 13 7 156125829 RNF32 GGGCGGCTGAGGAG CGTGGCTGCGCCCA CAAAGCCG cg08097417 335.8053 7.62E-203 KLF14 7 130419133 TGTTTAACAGCCTC 14 7 130069673 AGAAATTATCTTGT CTCCGCGTTCTTTC TTCTGCCG cg24079702 180.3007 4.70E-198 FHL2 2 106015771 CGCCCGAGAGCCCA 15 2 105382203 CGCCGGAGGCGACG CTCCCGATAACTAC TGTGCTCC cg02650266 274.1478 3.09E-191 4 147558239 GCTGTCCTCAGGAG 16 4 147777689 CCGCCAGAGTGCTG GGGAAGGCGGCAGC AACGAGCG cg06493994 327.7177 2.52E-189 SCGN 6 25652602 AAGAAATACGGTGA 17 6 25760581 AGGAGTCCTTCCCA AAGTTGTCTAGGTC CTTCCGCG cg16419235 299.9665 2.12E-185 PENK 8 57360613 CAAAGGGCTGATTT 18 8 57523167 CTACAGTCGCTAGG ACCTGCAGCGGCGC TGCTCCCG cg22736354 254.184 6.70E-185 NHLRC1 6 18122719 CTCGAGTGCAAGGT 19 6 18230698 GTGCTTTGAGAAGT TTGGCCACCGGCAG CAGCGGCG cg07547549 192.8751 2.24E-183 SLC12A5 20 44658225 GCTCAGCTCCATTG 20 20 44091632 GAATGCTCCGGGCG CTGTCCAAGGTGCT GGAATGCG cg21572722 229.9224 9.15E-183 ELOVL2 6 11044894 CGGAGCCCCAGGAA 21 6 11152880 TACCCACCCGCTGC CCAGATCGGCAGCC GCTGCTGC cg04400972 288.3508 1.18E-181 TRIM45 1 117665053 CGGTCTCCCGAACC 22 1 117466576 GGTCCCCGTAACGC GAGCCTGAGATGCC CTCACCCC cg26290632 273.449 2.57E-177 CALB1 8 91094847 CATCACAGCCTCAC 23 8 91164023 AGTTTTTCGAGATC TGGCTCCATTTCGA CGCTGACG cg21296230 270.4638 2.37E-176 GREM1 15 33010536 GCGGGGGTGAATTG 24 15 30797828 TGAAGAACCATCGC GGGGTCCTTCCTGC TGAGGCCG cg25778262 243.1574 1.21E-174 CPM 12 69327449 TAGCCTCGCTGGGC 25 12 67613716 AGCTTGGCACTGCT GGGAGCTTGGCTCG CCCTGCCG cg13649056 307.4308 3.80E-172 9 136474626 GGGGGATGCCGGGA 26 9 135464447 GCGGCCTGGGGAGC TGTCCCTGGTGCTG ACGGCTCG cg00748589 267.9466 2.85E-171 12 11653486 GCTCTACCTCAAGG 27 12 11544753 AGCTCAGGGCCATC GTGCTGAACCAACA GAGGCTCG cg23500537 227.7268 4.82E-170 5 140419819 GCAGCCACACATCC 28 5 140400003 AAGGCTGACAGGGC GGGCACTCTGCCAA GTCCTGCG cg03607117 451.6398 1.76E-169 SFMBT1 3 53080440 CGCCCTGGCCCAGC 29 3 53055480 CCCGATCCAGCCTG CGCCTCACCTCGGG TTGTAGAC cg23091758 311.9045 7.86E-169 NRIP3 11 9025767 GGAGGCGGCGGCGC 30 11 8982343 TGGTGGGGACTGAC CCGGCAGTCCGAGA ATCCACCG cg07955995 425.3423 5.65E-168 KLF14 7 130419159 CGCTCTGTTACCAT 31 7 130069699 TACCTGGCTCGCCG GCAGAAGAAAGAAC GCGGAGAC cg04836038 405.8761 6.08E-166 DOCK9 13 99739382 AGAGGTCTCAGGAA 32 13 98537383 AGTAGCCTTTATTT ATGTGGCACCGATC GGAACCCG cg20426994 414.7314 8.35E-165 KLF14 7 130418924 GTGGCGCTTGGCAG 33 7 130068864 CAGGTGTGACAGAC CTCCTCCGGGGCGC CTGATCCG cg08128734 -153.801 4.55E-164 RASSF5 1 206685423 CGGGGCTAAATCAA 34 1 204752046 GGAAAACACACGCT ACACACTCAGTGCT GCTGGGTG cg24436906 272.7476 2.07E-162 BOK 2 242498081 CGGGGAAGCTCGGA 35 2 242146754 AAGCGTCTCCCCGA CTCCGCCCCCAGGG TTGCCTTT cg04908625 175.9083 1.42E-161 ADCY5 3 123166882 CGGCCGCGCGCCCC 36 3 124649572 TTGCCCCGCCGCTC CTCCAGACCCACCT CCACCGAG cg00481951 249.7016 2.89E-161 SST 3 187387650 GTTTCAGCACCTGG 37 3 188870344 GTCAGCGCTTCCCA GGGTCAGCACCAGG GATAGACG cg15108590 324.0472 1.75E-156 CBS 21 44494906 GTCTTGGGGAGCCC 38 21 43367975 GCGGGTTCGGGTCT GGGTCGCCTGGCGA GCTTTCCG cg22282410 284.9341 3.72E-154 PTPRN2 7 158380884 CCCGGTGCTGGGGG 39 7 158073645 TCGCACTGTCCCTG GGGACGGCGGGGGC CTAAGCCG cg21801378 403.3465 1.94E-153 BRUNOL6 15 72312125 CGGGCTAAACCCCG 40 15 70399179 GTCCCGCCGTACCC ATGAAGGACCACGA CGCCATCA cg04940570 264.1321 3.97E-152 TEAD1 11 12696758 ACACACCCTCGGGC 41 11 12653334 GCCTTGGACGGGGT GCGCTGGGGAGCCA GAAGTTCG cg04084157 463.0799 5.79E-150 VGF 7 100809049 AGCATTTCATTCAT 42 7 100595769 TCATTCATTCATTC ATTTCCCGGAGCTC CGCTAGCG cg25410668 202.9914 1.20E-148 RPA2 1 28241577 CACCGCGTGGAGTT 43 1 28114164 GCTTGTTCTTTTAC ATAGGAGGTCACAT TCTCTTCG cg04865692 240.2425 1.40E-144 KCNC3 19 50831762 GACGAGACCGACGT 44 19 55523574 GGAGGCCTGCTGCT GGATGACCTACCGG CAGCATCG cg04528819 344.067 2.76E-143 KLF14 7 130418315 CGCCCCGGAGGAGG 45 7 130068855 TCTGTCACACCTGC TGCCAAGCGCCACC AATGCCCC cg10804656 181.8587 3.81E-143 10 22623460 CGGATCCCGCCAAA 46 10 22663466 TTTGAACGCGAGAT TGTCAGGCCCTGAG GGGCTTGA cg09499629 498.3802 5.10E-143 KLF14 7 130419136 CCCAGAAGTTCCGA 47 7 130069676 CTGGGGAGTTTCGC TCTGTTACCATTAC CTGGCTCG cg03032497 226.3201 8.93E-143 14 61108227 ATCTAACTCAACCC 48 14 60177980 CTTTAGATATTCTT CCAGGTGGAATTAT TGGATTCG cg09401099 283.2574 3.24E-142 3 156534380 CGCGAAGGCCACTC 49 3 158017074 GCTGGCGACCCCTT CCCGGGTCTCCTAG CCCTGGCC cg12373771 276.5215 1.04E-140 CECR6 22 17601381 AGCACCAGTACAGG 50 22 15981381 TCGGTGACGGCGAT GAGGTACAGGTCCA GCAGGCCG cg07927379 516.3105 2.87E-140 C7orf13; 7 156433108 CGGCCCTCACTACA 51 7 156125869 RNF32 CGAGGCCTGGGCGC CTGCACGCCCCCGT GCTTCAGC cg18473521 176.2876 7.69E-138 HOXC4 12 54448265 TTACCCATTCTCGC 52 12 52734532 TCGTAAATCCAGTT CAATTGTGCTAACC CAGAGTCG cg07806886 370.4424 3.83E-137 STXBP5L 3 120626899 CGGCGCCAATCCTA 53 3 122109589 GATTCGATAGGGTA AGTTCTGTGGTCTC CAGGGCAG cg01528542 -196.924 6.73E-137 12 81468232 CGTTAACCTCTGCT 54 12 79992363 AGTGATGACCAAAC CTGGTAAAGATTGT AAAGTGGG cg03473532 -225.757 9.80E-137 MKLN1 7 131008743 CGTATGTGTTTGAG 55 7 130659283 ATAGCAGTTGTTTA CTATCACTTGAAAA TTCTGAAT cg25478614 251.6982 1.34E-134 SST 3 187387866 GGACCCAGAAAAGC 56 3 188870560 ACCAAAACTCTTTA GAAGGACTGAGCAT CCCTTACG cg21186299 812.41 2.75E-134 VGF 7 100808810 GCGACGGTCGAGGT 57 7 100595530 CTGGCGTCCCGTGG GCTGGGCTCAGCTG GGTCGGCG cg05093315 -242.236 6.14E-134 SAAL1 11 18127958 CGAGACCAGCCTGG 58 11 18084534 GCAACATAGATCAG AAGGCGAATAGAAT AAGTCCGC cg23441616 915.0279 3.75E-132 MYCBP2 13 77901383 GGGTTTGGGGCTGT 59 13 76799384 TGGGTTGTGCGGAA TCTGAAGTAGTCCA CTTCTCCG cg17321954 384.39 4.95E-132 STXBP5L 3 120626881 CGATAGGGTAAGTT 60 3 122109571 CTGTGGTCTCCAGG GCAGAAGAAATCTG TGGATAGG cg03771840 183.5214 1.73E-131 TRIM15 6 30140145 CGCCCTTCGCGCGC 61 6 30248124 CCCACTTCAGCCTT TCAGCGTAAGGCAG GAACCTTT cg03545227 347.1845 1.01E-130 PTPRN 2 220173100 AGGTCTAGTGGAGA 62 2 219881344 GTCCTCGCTCTGTG ACCCCTTCCTCTCT GGTAACCG cg18826637 -134.891 1.49E-130 2 145116633 TCCATTGGAAACTC 63 2 144833103 CCCTCTAAGCTGTG CATTTTTAGGCTGT GGTCATCG cg23186333 -163.283 2.02E-129 CD44 11 35161900 TTTCTTTGTCTATG 64 11 35118476 TATGTACAGATAAT TACATGGCCGATTT GCTTATCG cg06570224 230.2161 3.56E-129 3 157812475 AGCAGGGGAGATGG 65 3 159295169 TGGCTCCCTCTCGG GGCCAGTCTGCCCC AAGCAGCG cg13848598 196.8931 5.52E-129 ADRB1 10 115804578 GCAGGTACACGAAG 66 10 115794568 GCCATGATGCACAG GGGCACGTAGAAGG AGACTACG cg20482698 302.1472 3.25E-126 ACTN2 1 236849994 CCTCCTGGATCATG 67 1 234916617 TACTCATCCTCGTC GTACACGTAGTTGT ACTGCACG cg24430580 506.5864 1.97E-125 PITX2 4 111544235 CACCAGGAAGCCCG 68 4 111763684 CCTCTGGTTTTAAG ATGTTAGGCCAACA GGGAAGCG cg16181396 275.0637 2.22E-125 ZIC1 3 147126206 CTCTCTCTTGCGTT 69 3 148608896 ATTTTTCTGTTTTC TGCCTTTCCGTTGT CTCCTTCG cg23744638 -173.575 1.82E-124 11 10323902 CACGAAGCTTTGGG 70 11 10280478 GAGCACTCTAGCCC CTGCTACTCACCCA TGCAAGCG cg11806672 567.9303 1.87E-124 POU4F1 13 79176608 TGTGGTACGTGGCG 71 13 78074609 TCCGGCTTGAAAGG ATGGCTCTTGCCCT GGGACACG cg26005082 551.7073 2.46E-124 MIR7-3; 19 4769660 ACCGAAGGAGGAGA 72 19 4720660 C19orf30 ATGCTATTTATTTC AGCACCAAATATCC GGACAGCG cg09809672 -183.887 5.19E-124 EDARADD 1 236557682 TTCATCTAGAAGGT 73 1 234624305 TTGACTCTGGCCAG ACAACCAGCGAGCA TCTTCTCG cg22285878 513.3208 6.26E-124 KLF14 7 130413173 TCTTCTGCCGGCGA 74 7 130069713 GCCAGGTAATGGTA ACAGAGCGAAACTC CCCAGTCG cg08706258 1112.51 1.92E-122 WSB1 17 25621230 CGGAGTCAACCACA 75 17 22645357 GACAATAGACCCTG TACCCAGCCTCGCG CCTGCGGA cg07920503 316.5951 2.90E-122 FAM123A 13 25745406 GAGGAGCAGGACCC 76 13 24643406 ACGACGGACTTGCC GAGGTGCTGGTGCT GGAGAGCG cg01429360 617.0854 3.26E-122 IGF2BP3 7 23509546 CGGGCCCACCTGAA 77 7 23476071 AGCGCCTCGATGGC CTTGAGGGCCCAGC TCTCGTCC cg12865028 242.6626 3.26E-121 4 13526659 GGGCTCTCCGAAAC 78 4 13135757 AGGCCGGGAAAGCT GAAAGCACAGTGAC CTCCTTCG cg08957484 224.7044 9.76E-121 CCNI2 5 132083532 GGTCCTGGGCCAGC 79 5 132111431 TGCAAGTGGCAGAG CAGCCGGCGCTCGT CCAGGTCG cg17621438 -217.539 2.47E-120 RNF180 5 63461216 CTGGCAACGCTACC 80 5 63496972 TGGGTTTAGTTTTC CCTTCGTATATCAC TATCTTCG cg18633600 232.9342 3.43E-119 LRTM2; 12 1940452 GGGCAACTGGGCCA 81 12 1810713 CACNA2D4 GGCCGTTGATGGAC AGGTCCAGGTGGCG GAGCAGCG cg18573383 394.9647 4.31E-118 KCNC2 12 75603401 GTGGAGACTGGCCG 82 12 73889668 CAGGTCAGGAGAGC TCACCACTTGAAGG TGAAGTCG cg10039299 305.1925 5.87E-117 2 96192273 GCAGTCCCTGAGCC 83 2 95556000 TCTGCAGGCAGTTC TTGGAGCCCTCGGG CTTTTGCG cg17101296 202.8553 1.51E-116 8 145925708 TGGGACAAGGACAG 84 8 145896517 GTCAGCGGGTCACA GGCCGGAAGTGAGA CTCGCCCG cg08540945 261.6127 1.61E-116 7 152591698 CGCGCTCCGCCCTT 85 7 152222631 TGCCTGCAGAGCGC TGGGGGTTTAAAGT CCTGAACC cg02561482 286.9963 1.88E-116 TFAP2B 6 50813551 CGGCAGCCCCTCCA 86 6 50921510 GCGGCTGATTCTAT GTCCTCAACACGAC TGGGCGCC cg26842024 466.3294 2.91E-116 KLF2 19 16436122 CAACAGCGTGCTGG 87 19 16297122 ACTTCATCCTGTCC ATGGGGCTGGATGG CCTGGGCG cg16969368 216.3395 2.96E-116 DHX40 17 57642752 TGCAGAGACCACTG 88 17 54997534 TGGCGTTGAAAAGA GGTGTCGTCGCGAC CTTCGGCG cg15626285 -186.581 7.21E-116 C1S 12 7167781 CGATTGCTTAATGC 89 12 7038042 TATTTTTCAGCCAA AGGGTGTGTTTCTG AGTTTTCG cg19470159 426.8016 1.15E-115 C3orf50 3 167967842 CGCTTGGAGAGAGC 90 3 169450536 AGACAACAGTATGC CCCGCCCCACCTCG GACCTGGT cg23361092 345.3453 1.95E-115 13 79170923 CGGAGAGTTCTGGA 91 13 78068924 AATAAAATGAATTA TAACAAGGAGCTAA TTAAAAAC cg03763391 1044.918 5.91E-115 BUB1B 15 40453091 AGACAGCACCTGGG 92 15 38240383 GGTATTTGTTTTGC CTAAGCCTGCTGCA CTTCCACG cg03664992 406.7318 1.15E-114 BMP8A 1 39957393 GAGGCCGGGGCTGT 93 1 39729980 TCTGAGGGCTGGGA CTGTCAGCCAATCC GTCTGTCG cg08483876 338.5102 1.54E-114 8 145910754 AGGGCAGGGACACA 94 8 145881562 ACTCACTCTGGACA GGGTACAGTCACAC CCACTTCG cg18035229 381.9429 4.15E-114 PRDM14 8 70954270 TCTGTGAATGTGAA 95 8 71146824 TGGAACTAAGCGTT CCTTTCTCTCCCTC AATGGCCG cg00664406 164.0497 7.58E-114 GRM2 3 51740875 CGGGGATTCAGCAC 96 3 51715915 CACGAGGCGGACAG CTCCAGGCCCTGAG GTCCCCAG cg26720338 740.0267 6.22E-113 JPH3 16 87635575 ACCACAGGTGGTTT 97 16 86193076 TCTCCGGTGACAAA CAATGCTTCCTTCT TCCTTCCG cg11052516 804.1625 8.10E-113 LOC645323 5 87957175 CCTTGCAAGGCGGC 98 5 87992931 TGCTAAGCCTGGCT AATTTTAGATCTCC AGAATGCG cg07544187 245.416 1.67E-112 CILP2 19 19651235 CGCGTGGCCGCCGC 99 19 19512235 TGCTCCAACTACCA CGTGCGCTTCCGCT GCCCACTA cg19674669 448.8857 1.15E-111 GLB1L3 11 134146910 CGGTGCCCAGCCGC 100 11 133652120 TGGAGCCCCTGGCC TGCGTGCCCCACCC TGATTTTC cg07850154 -199.617 1.95E-111 RNF180 5 63461232 CGAAGGGAAAACTA 101 5 63496988 AACCCAGGTAGCGT TGCCAGCTTAAAAG TCCTAGGC cg07593137 -189.624 3.62E-111 CHMP4C 8 82644012 CAGCCCCATTTAAG 102 8 82806567 GTTTTTGATACACT GAGGATCATTCAGA AAACTTCG cg25148589 265.0785 9.11E-111 GRIA2 4 158141936 CGGCAGCTCCGCTG 103 4 158361386 AAAACTGCATTCAG CCAGTCCTCCGGAC TTCTGGAG cg07178825 214.9746 9.65E-111 TP73 1 3649574 CGACCTGCCCGACT 104 1 3639434 GCAAGGCCCGCAAG CAGCCCATCAAGGA GGAGTTCA cg10172783 341.995 1.02E-110 NAGS 17 42082036 CGCCATGACGACAA 105 17 39437562 CCAACTCTTGCCCC CCAAGAGTGGCAGT CTGTCTGG cg16247183 247.7465 2.90E-110 1 225865110 CGCTAGCGCCTCGG 106 1 223931733 TTACAGCCTTTCCC GCAAGGCTTCATTC AGTCGCGC cg03696327 555.4321 7.31E-110 GPR88 1 101005121 CACGATGCCCAGGT 107 1 100777709 AGCAGTGCAGCAGC AGAGCTGTCTGCGC CAGCAGCG cg05675373 346.861 1.37E-109 KCNC4 1 110754257 AGGCGGGTTCCCGG 108 1 110555780 TAGGGTGCGCAGGG TGCTGCGGTAGGTC TCATGTCG cg14044057 907.4472 2.19E-109 SPDYA 2 29033296 CGTGTGAATACGGT 109 2 28886800 GGCTTCTTGTGAGA AGGGGCCATTCTAT TGTAACTG cg01644850 996.0475 4.72E-109 ZNF551 19 58193231 CGGAGCTCTTCGGA 110 19 62885043 GTGTGTCCACTGCT TTGACCTCTGCGAA CTTGTATT cg13636189 622.6708 1.09E-108 NR4A3 9 102587074 CGCAGCTCAGCAGG 111 9 101626895 CCTCAGGGAAGGAA CTGGGTGCCCAAAC TCCGGCCT cg21870884 252.246 2.30E-108 GPR25 1 200842429 GCTGGTGGATACCT 112 1 199109052 TCGTGCTGCACCTG GCGGCAGCTGACCT GGGCTTCG cg19392831 244.0071 1.26E-107 PRLHR 10 120355756 CGGCCAAGCCAAAG 113 10 120345846 GCAGGAGTCAGCAC CACGGACAGCTTCC GCTGGATC cg21255438 330.2059 1.96E-107 PRDM14 8 70983760 CGGGGAGAAAAAAA 114 8 71146314 CCGAACACGTGTGC TACCCAGGGCCCCC AGATAAGC cg26496307 497.0731 2.89E-107 ZNF813 19 53970803 CGGCCAGTAAGGTT 115 19 58662615 GAGGCACTATTCAA AAGCCCTGGAATTG TCTGGAAC ch.1. 603.477 3.81E-107 DHX9 1 182831125 CAGGTAGGTGCTGA 116 1 181097747 3571292R TGAATTTGAGTGTG TTTAAATCTTAGAC TTACTGTA cg03399905 318.4912 4.54E-107 ANKRD34C 15 79576060 CCATGCTCGGCCTT 117 15 77363115 CTGGAAGATGCCCA CAGACACTGGCAAT AATGGACG cg24834740 1060.466 4.85E-107 PPP1R16B 20 37434552 CGCCCCGGCCCCCA 118 20 36867966 GCTAGGTGATAGCA GGCTGGGACCACCT CCCCGCCC cg02159381 438.4332 5.90E-107 BSX 11 122852523 AACACAGAGACCCA 119 11 122357733 ACCTACCCAGGAGC TTGTCTTCTTGCCT CTCCAGCG cg13782301 190.5049 7.31E-107 PRRT1 6 32116875 CGATGTATCCAAGT 120 6 32224853 CTGACGGCCCCAGA AACGGGTGTGCAGG GCGCCCAT cg16054275 -244.386 1.97E-106 F5 1 169556022 CGTCCGTTACCACT 121 1 167822646 GACCTGAGGCCTGC CTGGGTCCAAGCTC ACACTTGG cg18867659 694.0629 1.22E-105 NETO2 16 47178357 GGTCAAAACTTTGC 122 16 45735858 CCAGCTCAGCCTTG CTCGACCCTGGGCA GGGAAGCG cg22796704 -184.335 1.84E-105 ARHGAP22 10 49673534 CGACCACACCAGGC 123 10 49343540 ACCCAGGAGCAAGT GCTTTGAAATGCGG CTTTCTCC cg22158769 401.3016 2.00E-105 LOC375196; 2 39187539 ACGCGGGAACTCTT 124 2 39041043 LOC100271715 TGAGAGAGCGGCTC AGCGGCTTGGCCTT GCCGTGCG cg08858751 776.6992 2.31E-105 ZNF599 19 35264235 TCCGTCCCTTGTAG 125 19 39956075 CACTGCCTTCTGGG TAATGTAGTTTGAC GGAATCCG cg12052661 253.4845 2.47E-105 CACNA1B 9 140772545 GTGAAGCAGTTCTG 126 9 139892366 CTTGACCGGGATGG GGTTGTACAGCGCC ATGGTCCG cg11176990 422.1135 4.75E-105 LOC375196; 2 39187533 GAACTCTTTGAGAG 127 2 39041037 LOC100271715 AGCGGCTCAGCGGC TTGGCCTTGCCGTG CGCCTGCG cg10328877 482.2751 7.70E-105 MEIS2 15 37391187 CGCCGCCTAGACTA 128 15 35178479 CTAGCCTGGGCTGC TTGTTTTGTCTCTG AAATTGAC cg19855470 300.7657 1.03E-104 CACNA11 22 40060836 CAGCAGCTGGAACG 129 22 38390782 TGCTGGATGGCTTT CTTGTCTTCGTGTC CATCATCG cg12921750 479.2745 2.73E-104 NETO1 18 70535336 AGCCCCAAGCCATG 130 18 68686316 ACTAAGGAGCCCAT TTGGTAACTCTGCC CTCTTCCG cg02286549 1008.269 3.07E-104 TFEB 6 41700710 CGGTTTCTGCAGGC 131 6 41808688 AACAGGGGTTTCCC CAACCACAGCTGTC ATGAAAAC cg09240095 825.5141 3.45E-104 KCNMB4 12 70759304 CGGTGACCCTTGTG 132 12 69045571 GCAACTTAGGTCTC TGGCAGCCGAGTTG ACCCCAAC cg18760621 773.2701 3.64E-104 1 158083299 AAGGCAAATTGCCT 133 1 156349923 GCCTCGTGCATAAT AAGCCAGGCGTGGA GAGCAGCG cg20199655 866.8009 3.94E-104 KRAS 12 25404314 TGAGGGTGGCGGGG 134 12 25295581 TGCTCTTCGCAGCT TCTCTGTGGAGACC GGTCAGCG cg13464448 431.1235 2.04E-103 ADAMTS8 11 130297513 CCACGAGTAGGACC 135 11 129802723 AAGCGGTTTGTGTC TGAGGCGCGCTTCG TGGAGACG cg01974375 -337.238 2.70E-103 PI4KB 1 151298954 CGAGGGAGGTGTCA 136 1 149565578 AAGTTGGAAATCCT GAATGGGAAGGGCA CTGTCAAA cg24809973 205.9183 2.93E-103 8 72468820 TGAGAGCTGGGAAC 137 8 72631374 CTGCGCCAGTGACT GCGCGACAGTGTTG ACGGGCCG cg10850791 237.5174 8.50E-103 PABPC4L 4 135122718 CGGGCCAAGGGCGT 138 4 135342168 CCTGAAGACCTAGG GGGCCCCTCCGACC TCCCGACC cg24452260 266.3675 1.02E-102 GRIA2 4 158143538 TCGCGAGCTCCATG 139 4 158362988 TTCTCCTCTTTGGG ACAAGTTGTTGAAA TGGTTCCG cg02328239 308.6597 1.13E-102 GDNF 5 37837463 ACCAAGCTCTGCTC 140 5 37873220 CTCAAGTGACGGGG GCTCTGCTCTGCCA GGTGACCG cg18445088 250.5134 2.66E-102 CACNA1I 22 40081812 CGGGCAGCCTGCAG 141 22 38411758 ACCACGCTCGAGGA CAGCCTGACCCTGA GCGACAGC cg15121420 -267.283 5.74E-102 RAB17 2 238490819 CGAGCCCTGAAGCT 142 2 238155558 GGAAAGCCAACGTG CTGGCTGGAGCCAG AAGAGCAG cg23355126 876.1891 7.97E-102 TMEM50B 21 34852107 CGGTTGCCTGGCGC 143 21 33773977 CGGAGACCCACAGA CAGGACTCACCCAG CTTCCTCA cg20209308 340.6068 8.36E-102 GSC2 22 19137306 CGCCACCGCACCAT 144 22 17517306 CTTCAGCGAAGAGC AGCTGCAGGCGCTC GAGGCGCT cg20676716 270.1375 1.92E-101 HOXD1 2 177053568 GGCCCGAACCATGA 145 2 176761814 GCTCCTACCTGGAG TACGTGTCATGCAG CAGCAGCG cg11873482 306.5791 2.16E-101 TAC1 7 97361244 CGTCGATGCCCATA 146 7 97199180 ACATCTGGACCCAA TTGGGTTCTAAATG ACGCAATT cg09226692 566.8713 2.18E-101 DLK2 6 43422490 CGGACAGGCTGACC 147 6 43530468 GGGAGCCCCCAGAA TGCACAACAGGCAC ACGAGATG cg06475764 829.304 2.68E-101 NETO2 16 47177480 GGGAACATGGCCCT 148 16 45734981 GGAGCGGCTCTGCT CGGTCCTCAAAGGT AAGGACCG cg01820374 -284.307 4.33E-101 LAG3 12 6882083 TCCTGGGCTTGCTG 149 12 6752344 TTTCTGCAGCCGCT TTGGGTGGCTCCAG GTAAAACG cg22016779 -289.294 4.46E-101 DNER 2 230452311 CGTGGCCTGGTTAA 150 2 230160555 CCAATCTGTTGCAC TGGCTCCCTTTTAA GGGGCCTG cg19505546 239.4817 3.94E-100 5 139017263 GGGTGCAGAGGCCT 151 5 138997447 AGGGCGGGCAGGCC GGCAGACTGGGGTC GGGCCACG cg02830438 519.0376 7.14E-100 C14orf109; 14 93651416 CGGCGGAGCCTGCT 152 14 92721169 MOAP1 TGCAAAGCTGAGGT CCCGGATCTCACCT TCCTGTCC cg27569300 317.7173 9.75E-100 SYNM 15 99645065 CGCTGAGCCCGGCC 153 15 97462588 TGGCTAGCCCGCCA CCCCGCCCGCTGTT ACCCGACT cg00852549 528.7081 1.13E-99  NXPH1 7 8473457 TGGGCCCACAGGGA 154 7 8439982 CAAGTGGCTCCCGC GGTGTCTTCGGTGG CCGCAGCG cg03750778 988.2525 1.26E-99  DST 6 56708763 ATCTGCGGCTTTGT 155 6 56816722 TTCTCAGGCACCTG TTGTGGATCCCAAA TAGAAACG cg11847992 -141.98 2.35E-99  5 95590917 CGATGCTGCTTCAT 156 5 95616673 GATATGTGTCAAAA TAAATGCAGGAAAC AGCTTTTG cg17729667 314.0009 4.10E-99  NINL 20 25566382 GGCGGCTCTGGCCA 157 20 25514382 GTTTGGAGCCTGGG GTGACCCTTGGAGC TGACCTCG cg27067781 198.7199 4.35E-99  PRRT1 6 32116853 CGTCTCGCCTTGCG 158 6 32224831 AGCAAGCTCGGAAT CCAGTTCCTCAGGA ACCCCTCC cg08804013 965.0246 5.05E-99  NFAT5; 16 69600791 CGACGGCGCAAAAA 159 16 68158292 MIR1538; CAAGCTGGAAAGGG AGGAAAATGGTGAC CCTGCACT cg26158959 461.8299 9.67E-99  SYT14 1 210111162 TTCAACCAAGGAGA 160 1 208177785 CCTGTCCATGGTCC TGACCACATCATTT GCCACTCG cg00171565 685.6216 9.82E-99  PKM2; 15 72523739 CATTGGTCATCAGG 161 15 70310793 TTTCTTAAAATGTG ACTCTGAATCTGTG TCCTTCCG cg22059812 272.757 9.97E-99  HTR6 1 79992564 CCAGGCTGATGAGG 162 1 19865151 CAGAGGTTGAGGAT GGAGGCGCTGCAGC ACATCACG cg06998238 954.4673 1.10E-98  ZNF121 19 9695323 TTTCAGCCACATAG 163 19 9556323 GACCCAGTCAAACA CAGAAATTGTAGTT TCTTCCCG cg15500658 1024.487 1.17E-98  SPEN; 1 16174610 CATGGTCCGGGAAA 164 1 16047197 FLJ37453 CCAGGCATCTCTGG GTGGGCAACTTACC CGAGAACG cg26946259 340.1318 1.31E-98  2 119599545 CGGAGACCAGGCGT 165 2 119316015 GTCCCGCCAGACCC TTCAGACCCAGGCT AAACCCAA cg21166964 329.2406 2.15E-98  5 72529816 CGGGCAGGCTCAAA 166 5 72565572 AGAAAAAGAATAAT TAGGGATAATTGCT TGTGTCCA cg11693709 -160.409 2.40E-98  PAK6 15 40542019 GGCATTGGCAGGCC 167 15 38329311 AGTATGGTCTGGGA GGGCAGCAAGGTGG GCACATCG cg06369624 366.1836 4.10E-98  KCNS1 20 43727355 ACTCGCTCACAAAG 168 20 43160769 GTTTCAGTGCTCCT CCCTGCGGACACCA GAAGGGCG cg11436113 -208.203 4.67E-98  20 19191145 AATAGAAACCCAAG 169 20 19139145 AATCATTTCTGTGT GCCACAGGAGTGCT CTCCCCCG cg17039022 239.849 5.60E-98  ATP2B4 1 203595145 CGGCTAATGACAGA 170 1 201861768 GCCAACGATTCAAG ACCAAGTCAGACAG ACTCCAAA cg12543649 1192.837 7.98E-98  THBS3 1 155176868 CGTTGTGGACACCA 171 1 153443492 GGTGCCACTCCTGT GGGGGATCAGCACA GCATCTCC cg09729848 723.7335 8.76E-98  ADAMTS2 5 178770998 CGAGGAGGAGCCTG 172 5 178703604 GCAGTCACCTCTTC TACAATGTCACGGT CTTTGGCC cg10833014 887.0278 1.09E-97  WDR20; 14 102605952 CGGTCAACTAGACC 173 14 101675705 HSP90AA1 CCACTAGCTGAAGC CGGCATCACCTGGG AAGCAGCC cg01844642 214.6266 1.62E-97  GPR62 3 51989764 GGGGTTGATCCTGG 174 3 51964804 CAGCTGTCGTGGAG GTGGGGGCACTGCT GGGCAACG cg25321549 823.9165 1.77E-97  ZSWIM6 5 60629121 CATCCTGGAGGGCT 175 5 60664878 GTTCGCCGGTTTCG GGGGTGGATGTGGA CAAAGGCG cg08385097 915.1697 2.09E-97  PAPOLG 2 60984209 CGGGAACTGTTTCT 176 2 60837713 GACTTATCAAAGTG TGAACAAGAGGTAC AGACCGGT cg23032032 387.8131 2.33E-97  FOXA1 14 38064513 CGAGGAGGTGGGCA 177 14 37134264 CTCAAGCGACGTAA GATCCACATCAGCT CAACTGCA cg17887993 738.1113 4.68E-97  MATN4 20 43922449 CGCGCCTGGAGGAT 178 20 43355863 CTGGAGAACCAGCT GGCCAACCAGAAGT GAGGGCCA cg26456957 664.7293 4.78E-97  PPP1R12C 19 55629363 GATGAATAGCAGAC 179 19 60321175 TGCCCCGGGGCAGT TAGGAATTCGACTG GACAGCCG cg04588840 348.6705 5.42E-97  CPXM1 20 2781685 CTTGATGTCAGCAA 180 20 2729685 AGTTTGCACAATGG GTCTTAACGTGCAC TCATTCCG cg17412974 -326.896 5.69E-97  12 80496965 TGCTGACCTTCGTA 181 12 79021096 GTGTCCTCGTACAA CCTGAACTTCATCG TCCTTTCG cg07399288 1080.87 6.16E-97  PMS2L4; 7 66767504 CTGGGCTCCCATTG 182 7 66404939 STAG3L4 GCTGCTTTTGACGT TGTGCTCCACCCTT TCTGGGCG cg26931990 241.6339 6.27E-97  IFT140 16 1661230 CGGCCGCCAGCTGC 183 16 1601231 TTTCTTGGGGGCGC TCCCTGCCTCGCTT GGCTCTGT cg19049194 306.1541 2.12E-96  2 175193754 TTTATCTAGAAAAC 184 2 174902000 TTTTCAAGCAAAGA CAAGGTCCTCTCGG CTTGTCCG cg08677617 246.9861 2.32E-96  10 102484048 TGTTGAGAGCGATT 185 10 102474038 TTAATTCTCATTCT GTACCTGCAGATGC CGCGGCCG cg05215004 663.8819 2.57E-96  LOC285780 6 6546556 CAAAGCAGATGACC 186 6 6491555 TGGCAGGAACCAGC CGCAGTGAAGCCAC CGCAACCG cg14022202 722.7556 3.78E-96  MTMR2 11 95656984 CTTCAGAAACCAGA 187 11 95296632 ATCCGCGAATTGGG GCAACAATCCAGCA GGTCCCCG cg21632975 263.091 8.60E-96  NOVA2 19 46456210 CGTCTACCTAGAGG 188 19 51148050 CAAAGACAGGAGAG AGGGAGTCCGTAAA ATCTGGAA cg09434500 332.8079 1.01E-95  GRIK5 19 42502897 GGGCTCCAGAGCCA 189 19 47194737 GGCCTCGGACTTCG CGGGGAACCAAAGG CAAAATCG cg09175724 764.3869 1.65E-95  CDC42EP2 11 65082792 CGGCCGCAGCTAAA 190 11 64839368 GATAGGAGAACAAC TCACTATCGGCTAA AAATACGG cg21300373 207.977 1.80E-95  4 165304540 TCAGCGCTAAACCC 191 4 165523990 AAGACAAAGGCTGC CCTGTGTCTTCCGT ACTCAGCG cg01897823 787.5659 2.02E-95  SOCS3 17 76356232 GCTCAGCCTTTCTC 192 17 73867827 TGCTGCGAGTAGTG ACTAAACATTACAA GAAGGCCG cg16076997 420.1993 3.41E-95  FOXD2 1 47905067 CGGGGCAGGGCAGA 193 1 47677654 GGCCTTCCTTCTCT ATAGACCACATCAT GGGCCACG cg15822346 603.9391 3.59E-95  SLC16A10 6 111408761 GGTGCGGGGCTGTG 194 6 111515454 ACCTAGAGGCTTCA GTGTCGATCCCCGA GGTGTTCG cg06121469 978.0709 7.94E-95  SPG11 15 44956098 CGGCCTGCTACGCT 195 15 42743390 AAGCTAGGCCTTCA AGCATGCCAGAGCA GTTAAGCA cg14513680 909.9088 9.49E-95  C9orf93 9 15552606 CCTGCTTTTTGAAA 196 9 15542606 CTGGTTCTTCTGCC CATCTTTAGAGCCA CAGCAACG cg03301331 303.8438 1.21E-94  RAB4A 1 229406681 CGGGACTCAGCCCC 197 1 227473304 CAACGCCCCCACCT GCCGCTCTGCCCAC CTCAGCGC cg02631838 279.3827 1.27E-94  HPCA 1 33358788 CGCCGCTCCAGGCC 198 1 33131375 CTCCACTGTCGGGC CCCGGTGTCCTCCA ACATCTCT cg14408969 913.5962 1.28E-94  C8orf40 8 42396118 ATAGCATCCTGGCC 199 8 42515275 ATATCCAGTTTTGA AAACACTACGGTGT CAGCCACG

TABLE 1B Marker UCSC_RefGene_Name UCSC_RefGene Accession UCSC_RefGene Group UCSC_CpG_Islands_Name HMM_Island Regulatory_Feature_Name cg16867657 ELOVL2 NM_017770 TSS1500 chr6: 11043913-11045206 6: 11151611-11153237 6: 11044102-11044892 cg10501210 1: 206063625-206063801 cg22454769 FHL2 NM_001039492; TSS200; TSS200; chr2: 106014878-106015884 2: 105381311-105382817 2: 106014507-106016259 NM_001450; NM_201557; 5′UTR; TSS200 NM_201555 cg04875128 OTUD7A NM_130901 Body chr15: 31775540-31776988 15: 29562601-29564280 cg24724428 ELOVL2 NM_017770 TSS1500 chr6: 11043913-11045206 6: 11151611-11153237 6: 11044102-11044892 cg06639320 FHL2 NM_001039492; TSS200; TSS200; chr2: 106014878-106015884 2: 105381311-105382817 2: 106014507-106016259 NM_001450; NM_201557; 5′UTR; TSS200 NM_201555 cg14556683 EPHX3 NM_024794; NM_001142886 1stExon; Body chr19: 15342626-15343181 19: 15203635-15204238 19: 15341951-15343455 cg23606718 FAM123C NM_152698; NM_001105194; 5′UTR; 5′UTR; 1st chr2: 131513363-131514183 2: 131229834-131230653 2: 131513688-131513993 NM_001105195; NM_001105194; Exon; 1stExon; 5′UTR; NM_001105193; NM_001105195 5′UTR cg07553761 TRIM59 NM_173084 TSS1500 chr3: 160167184-160168200 3: 161649892-161650878 3: 160166409-160168278 cg14361627 KLF14 NM_138693 TSS1500 chr7: 130417912-130419378 7: 130068467-130069793 7: 130418325-130419878 cg14692377 SLC6A4 NM_001045; NM_001045 1stExon; 5′UTR chr17: 28562387-28563186 17: 25586344-25587312 17: 28562266-28563419 cg19283806 CCDC102B NM_001093729 5′UTR 18: 66388995-66389733 cg00292135 C7orf13; NR_026865; NM_030936 Body; TSS1500 chr7: 156432433-156433670 7: 156125195-156126707 7: 156432754-156434135 RNF32 cg08097417 KLF14 NM_138693 TSS1500 chr7: 130417912-130419378 7: 130068467-130069793 7: 130418325-130419878 cg24079702 FHL2 NM_001039492; TSS200; TSS200; chr2: 106014878-106015884 2: 105381311-105382817 2: 106014507-106016259 NM_001450; NM_201557; 5′UTR; TSS200 NM_201555 cg02650266 chr4: 147558231-147558583 4: 147777501-147778016 4: 147557996-147558356 cg06493994 SCGN NM_006998; NM_006998 1stExon; 5′UTR chr6: 25652380-25652709 6: 25760360-25760750 6: 25652510-25652746 cg16419235 PENK NM_001135690 TSS1500 chr8: 57360585-57360815 8: 57522950-57523369 8: 57360377-57362115 cg22736354 NHLRC1 NM_198586 1stExon chr6: 18122250-18122994 6: 18230230-18231229 6: 18122473-18123542 cg07547549 SLC12A5 NM_020708; NM_001134771 Body; Body chr20: 44657463-44659243 20: 44090882-44092713 20: 44657985-44658436 cg21572722 ELOVL2 NM_017770 TSS1500 chr6: 11043913-11045206 6: 11151611-11153237 cg04400972 TRIM45 NM_025188; NM_001145635 TSS1500; TSS1500 chr1: 117664180-117665148 1: 117465578-117466781 1: 117663907-117665512 cg26290632 CALB1 NM_004929 1stExon 8: 91163987-91164262 cg21296230 GREM1 NM_013372 5′UTR chr15: 33009530-33011696 15: 30796823-30799072 cg25778262 CPM NM_198320; NM_001005502; TSS1500; TSS1500; chr12: 69327021-69327532 12: 67612814-67613799 12: 69326064-69327911 NM_001874 5′UTR cg13649056 chr9: 136474170-136474748 9: 135463992-135464726 9: 136474269-136474939 cg00748589 chr12: 11653232-11653775 12: 11544500-11545229 12: 11653353-11654101 cg23500537 5: 140400003-140400154 cg03607117 SFMBT1 NM_001005159; TSS1500; TSS1500; chr3: 53078956-53081101 3: 53053856-53056190 NM_016329; NM_001005158 TSS1500 cg23091758 NRIP3 NM_020645 TSS200 chr11: 9025095-9026315 11: 8981699-8983012 cg07955995 KLF14 NM_138693 TSS1500 chr7: 130417912-130419378 7: 130068467-130069793 7: 130418325-130419878 cg04836038 DOCK9 NM_015296; NM_001130049 TSS1500; TSS1500 chr13: 99738331-99740225 13: 98535557-98538321 13: 99739202-99739439 cg20426994 KLF14 NM_138693 1stExon chr7: 130417912-130419378 7: 130068467-130069793 cg08128734 RASSF5 NM_182663; NM_182664 Body; Body chr1: 206680236-206681444 cg24436906 BOK NM_032515 TSS200 chr2: 242498013-242499274 2: 242146569-242147947 cg04908625 ADCY5 NM_183357 1stExon chr3: 123166218-123168567 3: 124648975-124651818 3: 123166803-123167158 cg00481951 SST NM_001048 Body chr3: 187387914-187388176 3: 188870246-188870359 cg15108590 CBS NM_000071 5′UTR chr21: 44494624-44496989 21: 43367599-43370089 cg22282410 PTPRN2 NM_130843; NM_130842; TSS1500; TSS1500; chr7: 158379328-158381221 7: 158072055-15807421.9 7: 158379935-158381567 NM_002847 TSS1500 cg21801378 BRUNOL6 NM_052840 1stExon chr15: 72611946-72612802 15: 70399042-70400040 15: 72611781-72613209 cg04940570 TEAD1 NM_021961 5′UTR chr11: 12695414-12696981 11: 12651991-12653557 11: 12695339-12696865 cg04084157 VGF NM_003378 TSS200 chr7: 100806279-100809064 7: 100594926-100596772 7: 100808711-100809141 cg25410668 RPA2 NM_002946 TSS1500 chr1: 28240584-28241535 1: 28113187-28114165 1: 28240552-28241702 cg04865692 KCNC3 NM_004977 1stExon chr19: 50831454-50832070 19: 55523267-55524969 19: 50831452-50833214 cg04528819 KLF14 NM_138693 1stExon chr7: 130417912-130419378 7: 130068467-130069793 cg10804656 chr10: 22623350-22625875 10: 22663357-22663769 cg09499629 KLF14 NM_138693 TSS1500 chr7: 130417912-130419378 7: 130068467-130069793 7: 130418325-130419878 cg03032497 chr14: 61108954-61109786 14: 60177929-60179820 cg09401099 chr3: 156533839-156535131 3: 158016534-158017978 cg12373771 CECR6 NM_031890; NM_001163079 1stExon; 5′UTR chr22: 17600563-17602611 22: 15980564-15982862 cg07927379 C7orf13; NR_026865; NM_030936 Body; TSS1500 chr7: 156432433-156433670 7: 156125195-156126707 7: 156432754-156434135 RNF32 cg18473521 HOXC4 NM_153633; NM_014620 Body; Body chr12: 54447744-54448091 12: 52734084-52734533 12: 54447856-54448358 cg07806886 STXBP5L NM_014980 TSS200 chr3: 120626880-120627579 3: 122109343-122110635 cg01528542 chr12: 81471569-81472119 cg03473532 MKLN1 NM_001145354 Body chr7: 131012460-131013190 7: 131008672-131009115 cg25478614 SST NM_001048 Body chr3: 187387914-187388176 3: 188870501-188870889 cg21186299 VGF NM_003378; NM_003378 1stExon; 5′UTR chr7: 100806279-100809064 7: 1.00594926-100596772 7: 100808711-100809141 cg05093315 SAAL1 NM_138421 TSS1500 chr11: 18127296-18127711 11: 18127220-18128173 cg23441616 MYCBP2 NM_015057 TSS1500 chr13: 77900504-77901140 13: 76798159-76799513 13: 77901146-77901558 cg17321954 STXBP5L NM_014980 TSS200 chr3: 120626880-120627579 3: 122109343-122110635 cg03771840 TRIM15 NM_033229 3′UTR chr6: 30139718-30140263 6: 30247613-30248242 6: 30137754-30140152 cg03545227 PTPRN NM_002846 Body chr2: 220173021-220173271 2: 219881281-219882527 2: 220172822-220173572 cg18826637 2: 145116478-145116676 cg23186333 CD44 NM_001001389; Body; Body; Body; chr11: 35160375-35161000 11: 35160307-35162010 NM_001001392; Body; Body NM_000610; NM_001001390; NM_001001391 cg06570224 chr3: 157812053-157812764 3: 159294712-159295751 cg13848598 ADRB1 NM_000684 1stExon chr10: 115803358-115805468 10: 115792700-115795458 cg20482698 ACTN2 NM_001103 1stExon chr1: 236849472-236850323 1: 234916096-234916946 1: 236849424-236850009 cg24430580 PITX2 NM_000325; NM_000325; 1stExon; 5′UTR; Body; chr4: 111542062-111544464 4: 111762274-111764019 4: 111544213-111544369 NM_153426; Body NM_153427 cg16181396 ZIC1 NM_003412 TSS1500 chr3: 147126988-147128999 3: 148608809-148608897 cg23744638 chr11: 10324353-10324828 cg11806672 POU4F1 NM_006237 Body chr13: 79175610-79177985 13: 78073612-78074696 cg26005082 MIR7-3; NR_029607; NR_027148 TSS1500; Body 19: 4720522-4720736 19: 4769500-4769890 C19orf30 cg09809672 EDARADD NM_080738; NM_145861; TSS1500; 5′UTR; 1stExon chr1: 236558459-236559336 NM_145861 cg22285878 KLF14 NM_138693 TSS1500 chr7: 130417912-130419378 7: 130068467-130069793 7: 130418325-130419878 cg08706258 WSB1 NM_015626; NM_134265; 5′UTR; 5′UTR; 1stExon; chr17: 25620999-25621730 17: 22645071-22645997 17: 25620827-25621911 NM_015626; 1stExon NM_134265 cg07920503 FAM123A NM_199138; NM_152704 1stExon; 1stExon chr13: 25743998-25746127 13: 24641999-24644089 13: 25745311-25745491 cg01429360 IGF2BP3 NM_006547 Body chr7: 23508184-23509712 7: 23474024-23476225 cg12765028 chr4: 13526553-13526770 4: 13133203-13135868 cg08957484 CCNI2 NM_001039780 1stExon chr5: 132082873-132083911 5: 132110589-132111953 5: 132082544-132084072 cg17621438 RNF180 NM_001113561; TSS1500; TSS1500 chr5: 63461448-63462106 NM_178532 cg18633600 LRTM2; NM_001163925; 12: 1810610-1810818 12: 1939931-1940497 CACNA2D4 NM_001039029; NM_172364; NM_001163926 cg18573383 KCNC2 NM_153748; NM_139137; 1stExon; 1stExon; chr12: 75601081-75601752 NM_153748; 5′UTR; 5′UTR; 1st NM_139137; Exon; 5′UTR NM_139136; NM_139136 cg10039299 chr2: 96192055-96193072 2: 95555724-95556799 2: 96191893-96192915 cg17101296 chr8: 145925410-145926101 8: 145895807-145896910 cg08540945 chr7: 152591458-152591706 7: 152222028-152222744 7: 152590901-152592150 cg02561482 TFAP2B NM_003221 3′UTR chr6: 50813314-50813699 6: 50921228-50921944 cg26842024 KLF2 NM_016270 Body chr19: 16435202-16438064 19: 16296270-16299051 cg16969368 DHX40 NM_001166301; TSS200; TSS200 chr17: 57642720-57643294 17: 54997503-54998169 17: 57642284-57643729 NM_024612 cg15626285 C1S NM_001734; NM_201442 TSS200; TSS200 cg19470159 C3orf50 NR_021485 Body chr3: 167967246-167968130 3: 169449281-169450798 3: 167967472-167967926 cg23361092 chr13: 79170114-79171231 cg03763391 BUB1B NM_001211 TSS200 chr15: 40453005-40453685 15: 38240321-38240977 15: 40452682-40453925 cg03664992 BMP8A NM_181809; NM_181809 1stExon; 5′UTR chr1: 39956424-39958137 1: 39728549-39730700 1: 39956370-39957859 cg08483876 chr8: 145909676-145912846 8: 145880426-145883921 cg18035229 PRDM14 NM_024504 TSS1500 chr8: 70981873-70984888 8: 71144880-71147746 cg00664406 GRM2 NM_000839; NM_001130063 TSS1500; TSS1500 chr3: 51740740-51741413 3: 51715881-51716416 3: 51740394-51741198 cg26720338 JPH3 NM_020655 TSS1500 chr16: 87636506-87637284 16: 86192682-86195809 cg11052516 LOC645323 NR_015436 Body chr5: 87956489-87957187 cg07544187 CILP2 NM_153221 Body chr19: 19650683-19651274 19: 19511515-19513041 cg19674669 GLB1L3 NM_001080407 Body chr11: 134145559-134147180 11: 133650782-133652625 cg07850154 RNF180 NM_001113561; TSS1500; TSS1500 chr5: 63461448-63462106 NM_178532 cg07583137 CHMP4C NM_152284 TSS1500 chr8: 82644603-82644849 cg25148589 GRIA2 NM_001083619; 1stExon; 5′UTR; 5′ chr4: 158143296-158144053 NM_000826; NM_001083620; UTR; 1stExon; 5′UTR NM_000826; NM_001083619 cg07178825 TP73 NM_001126240; Body; Body; 3′UTR; chr1: 3649294-3649674 1: 3639248-3639685 1: 3649524-3649611 NM_005427; NM_001126242; 3′UTR NM_001126241 cg10172783 NAGS; PYY NM_153006; NM_004160 1stExon; TSS200 chr17: 42082027-42084972 17: 39437458-39440004 cg16247183 chr1: 225865068-225865328 1: 223931692-223932027 cg03696327 GPR88 NM_022049 Body chr1: 101004471-101005885 1: 100777157-100778458 1: 101004217-101005756 cg05675373 KCNC4 NM_001039574; 1stExon; 1stExon; chr1: 110752256-110754794 1: 110553818-110556317 NM_004978; NM_153763 1stExon cg14044057 SPDYA NM_182756; NM_001142634 TSS1500; TSS1500 chr2: 29033351-29034011 2: 29033093-29034127 cg01644850 ZNF551 NM_138347 TSS200 chr19: 58193268-58193638 19: 62884977-62885628 19: 58192869-58194184 cg13636189 NR4A3 NM_173199; NM_173198; 5′UTR; 5′UTR; 5′UTR chr9: 102581791-102587561 9: 101625826-101627570 9: 102586760-102587409 NM_006981 cg21870884 GPR25 NM_005298 1stExon chr1: 200842196-200843388 1: 199108820-199110011 cg19392831 PRLHR NM_004248 TSS1500 chr10: 120353692-120355821 10: 120344980-120346127 10: 120355066-120355940 cg21255438 PRDM14 NM_024504 TSS200 chr8: 70981873-70984888 8: 71144880-71147746 cg26496307 ZNF813 NM_001004301 TSS200 chr19: 53970802-53971473 19: 58662500-58663285 19: 53970386-53971554 ch.1.3571292R DHX9 NM_001357 Body cg03399905 ANKRD34C NM_001146341 5′UTR chr15: 79576059-79576270 15: 77363046-77363443 cg24834740 PPP1R16B NM_015568 5′UTR chr20: 37434206-37435592 20: 36867542-36869198 20: 37434191-37434662 cg02159381 BSX NM_001098169 TSS200 chr11: 122852411-122852699 11: 122357622-122357909 11: 122852441-122852883 cg13782301 PRRT1 NM_030651 3′UTR chr6: 32116590-32117229 6: 32224481-32225389 6: 32116667-32116975 cg16054275 F5 NM_000130 TSS1500 1: 169555452-169556050 cg18867659 NETO2 NM_018092 TSS1500 chr16: 47176787-47178446 16: 45734289-45736098 16: 47177731-47178968 cg22796704 ARHGAP22 NM_021226 Body chr10: 49674243-49674776 cg22158769 LOC375196; NR_028386; NM_001145451 TSS200; Body chr2: 39186777-39187968 2: 39040222-39041697 2: 39187021-39187940 LOC100271715 cg08858751 ZNF599 NM_001007248 TSS200 chr19: 35263648-35264275 19: 39955442-39956076 19: 35263430-35264597 cg12052661 CACNA1B NM_000718 1stExon chr9: 140771300-140773513 9: 139891122-139893552 9: 140772183-140772743 cg11176990 LOC375196; NR_028386; NM_001145451 TSS200; Body chr2: 39186777-39187968 2: 39040222-39041697 2: 39187021-39187940 LOC100271715 cg10328877 MEIS2 NM_172316; NM_170674; 1stExon; Body; 5′UTR; chr15: 37392601-37392829 15: 35178347-35178799 15: 37390925-37391332 NM_002399; Body; 5′UTR; NM_170675; Body; TSS1500; Body NM_172316; NM_170677; NM_172315; NM_170676 cg19855470 CACNA1I; CACNA1I NM_001003406; Body; Body chr22: 40060601-40061031 22: 38389756-38390938 NM_021096 cg12921750 NETO1 NM_138966 TSS1500 chr18: 70533965-70536871 18: 68684946-68688303 18: 70535222-70535468 cg02286549 TFEB NM_001367827; 5′UTR; 5′UTR chr6: 41701881-41703481 6: 41700492-41700940 NM_007162 cg09240095 KCNMB4 NM_014505 TSS1500 chr12: 70759437-70761052 12: 69045232-69046021 12: 70759300-70759423 cg18760621 chr1: 158083270-158083540 1: 156349654-156350159 1: 158082972-158083710 cg20199655 KRAS NM_004985; NM_033360 TSS1500; TSS1500 12: 25294434-25295836 cg13464448 ADAMTS8 NM_007037 1stExon chr11: 130297401-130298517 11: 129802612-129803797 11: 130297323-130298140 cg01974375 PI4KB NM_002651 TSS1500 chr1: 151300522-151300724 1: 151298798-151298969 cg24809973 chr8: 72468560-72469561 8: 72631115-72632846 cg10850791 PABPC4L NM_001114734 5′UTR 4: 135341838-135342385 cg24452260 GRIA2 NM_001083619; Body; Body; Body chr4: 158143296-158144053 4: 158362127-158363368 NM_000826; NM_001083620 cg02328239 GDNF NM_000514 5′UTR chr5: 37836747-37840726 5: 37872149-37873835 cg18445088 CACNA1I NM_001003406; Body; Body chr22: 40081519-40082390 22: 38411527-38412481 22: 40081445-40082681 NM_021096 cg15121420 RAB17 NM_022449 Body 2: 238490196-238490845 cg23355126 TMEM50B NM_006134 5′UTR chr21: 34851229-34852702 21: 33773438-33774743 21: 34852040-34852861 cg20209308 GSC2 NM_005315 Body chr22: 19136293-19338512 22: 17516155-17518857 22: 19136359-19137652 cg20676716 HOXD1 NM_024501 1stExon chr2: 177052957-177054350 2: 176761016-176762831 2: 177053532-177054285 cg11873482 TAC1 NM_013998; NM_013997; TSS200; TSS200; chr7: 97361132-97363018 7: 97199050-97199704 NM_013996; TSS200; TSS200 NM_003182 cg09226692 DLK2 NM_206539; NM_023932 Body; Body chr6: 43422368-43423705 6: 43530362-43531683 6: 43421555-43422964 cg06475764 NETO2 NM_018092 Body chr16: 47176787-47378446 16: 45734289-45736098 16: 47177261-47177605 cg01820374 LAG3 NM_002286 Body chr12: 6882855-6883184 12: 6881253-6882742 cg22016779 DNER NM_139072 Body 2: 230451331-230452578 cg19505546 chr5: 139017133-139017668 5: 138997178-138998057 5: 139017085-139017489 cg02830438 C14orf109; NM_015676; NM_001098621; 5′UTR; 5′UTR; 1st chr14: 93650745-93651652 14: 92720388-92721575 14: 93650342-93652057 MOAP1 NM_001098621; Exon; TSS200 NM_022151 cg27569300 SYNM NM_145728; NM_015286 TSS1500; TSS1500 chr15: 99645030-99646444 15: 97462554-97464153 cg00852549 NXPH1 NM_152745 TSS200 chr7: 8473139-8475199 7: 8439680-8442368 cg03750778 DST NM_001144770; Body; Body; TSS1500; chr6: 56708059-56709166 6: 56815609-56817067 6: 56707727-56709327 NM_001144771; Body NM_183380; NM_001144769 cg11847992 cg17729667 NINL NM_025176 TSS1500 chr20: 25565437-25566547 20: 25513460-25514516 20: 25565222-25566520 cg27067781 PRRT1 NM_030651 3′UTR chr6: 32116590-32117229 6: 32224481-32225389 6: 32116667-32116975 cg08804013 NFAT5 NM_138714; NM_001113178; 5′UTR; Body; Body; chr16: 69599437-69600736 16: 68156939-68158333 16: 69600528-69600817 NM_138713; 5′UTR; TSS1500; NM_173214; Body NR_031719; NM_006599 cg26158959 SYT14 NM_001146261; TSS1500; TSS1500; chr1: 210111179-210112054 1: 210110983-210111308 NR_027458; NR_027459; TSS1500; TSS1500; NM_001146264; TSS1500; TSS1500 NM_153262; NM_001146262 cg00171565 PKM2 NM_002654; NM_182470; TSS200; TSS200; chr15: 72522131-72524238 15: 70309363-70311340 15: 72523315-72523809 NM_182471 TSS200 cg22059812 HTR6 NM_000871 1stExon chr1: 19991146-19992788 1: 19863734-19865375 cg06998238 ZNF121 NM_001008727 TSS200 chr19: 9694921-9695433 19: 9555900-9556398 19: 9694602-9695488 cg15500658 SPEN; NM_015001; NR_024279 1stExon; Body chr1: 16173889-16175396 1: 16046263-16047983 1: 16173682-16176432 FLJ37453 cg26946259 chr2: 119599458-119600966 2: 119315907-119316219 cg21166964 chr5: 72529099-72529976 5: 72564856-72565732 cg11693709 PAK6 NM_020168; NM_001128628; 5′UTR; 5′UTR; 5′UTR chr15: 40544352-40545512 NM_001128629 cg06369624 KCNS1 NM_002251 Body chr20: 43726297-43727372 20: 43726268-43727871 cg11436113 chr20: 19192459-19193902 cg17039022 ATP2B4 NM_001001396; TSS1500; TSS1500 chr1: 203598471-203598853 1: 203594755-203596253 NM_001684 cg12543649 THBS3 NM_007112 Body chr1: 155178547-155178980 1: 155175976-155177609 cg09729848 ADAMTS2 NM_021599; NM_014244 Body; Body chr5: 178770724-178772794 5: 178703118-178705392 5: 178769342-178771312 cg10833014 WDR20; NM_181291; NM_181308; TSS1500; TSS1500; chr14: 102605597-102606977 14: 101675134-101676861 14: 102605541-102606369 HSP90AA1 NM_001017963; 1stExon; 5′UTR; NM_001017963; TSS1500; TSS1500 NM_144574; NM_381302 cg01844642 GPR62 NM_080865 1stExon chr3: 51989763-51990639 3: 51964804-51965628 cg25321549 ZSWIM6 NM_020928 Body chr5: 60626505-60629809 5: 60661968-60665553 cg08385097 PAPOLG NM_022894 Body chr2: 60983193-60983870 2: 60982720-60984542 cg23032032 FOXA1 NM_004496 TSS200 chr14: 38063663-38065665 14: 37133439-37134763 cg17887993 MATN4 NM_030592; NM_030590; Body; Body; Body chr20: 43921949-43922642 20: 43355572-43356029 20: 43921750-43923312 NM_003833 cg26456957 PPP1R12C NM_017607 TSS1500 chr19: 55628488-55629105 19: 60320109-60321178 19: 55628884-55629492 cg04588840 CPXM1 NM_019609 TSS1500 chr20: 2780978-2781497 20: 2780246-2781714 cg17412974 12: 79021088-79021218 cg07399288 PMS2L4; NR_022007; NM_022906 TSS200; TSS200 chr7: 66767145-66768031 7: 66404594-66405450 7: 66766960-66768186 STAG3L4 cg26931990 IFT140 NM_014714 5′UTR chr16: 1660054-1665095 16: 1659488-1661475 cg19049194 chr2: 175193398-175193764 2: 174901271-174902076 cg08677617 chr10: 102484200-102484476 10: 102474032-102474107 cg05215004 LOC285780 NR_026970 Body chr6: 6546370-6547230 6: 6491370-6492312 6: 6546161-6548100 cg14022202 MTMR2 NM_201281; NM_016156; 5′UTR; Body; Body; chr11: 95656912-95657365 11: 95296479-95297009 11: 95656229-95657555 NR_023356; 5′UTR NM_201278 cg21632975 NOVA2 NM_002516 Body chr19: 46456209-46456503 19: 51147814-51148279 cg09434500 GRIK5 NM_002088 3′UTR chr19: 42502730-42503484 19: 47194629-47195338 19: 42500888-42503553 cg09175724 CDC42EP2 NM_006779 5′UTR chr11: 65081937-65083333 11: 64838535-64839930 11: 65081771-65083639 cg21300373 NM_001166373 TSS200 chr4: 165304328-165305177 4: 165523779-165524912 cg01897823 SOCS3 NM_003955 TSS200 chr17: 76354818-76357038 17: 73866128-73868633 17: 76356011-76356507 cg16076997 FOXD2 NM_004474 1stExon chr1: 47902793-47905518 1: 47675329-47678200 cg15822346 SLC16A10 NM_018593 TSS200 chr6: 111408426-111409484 6: 111515073-111516544 6: 111408087-111409949 cg06121469 SPG11 NM_025137; NM_001160227 TSS1500; TSS1500 chr15: 44955291-44955983 15: 44954821-44956641 cg14513680 C9orf93 NM_173550 TSS1500 chr9: 15552733-15553334 9: 15552576-15553107 cg03301331 RAB4A NM_004578 TSS200 chr1: 229406646-229407129 1: 227473083-227474029 1: 229406323-229407948 cg02631838 HPCA NM_002143 Body chr1: 33358469-33359449 1: 33131039-33132010 1: 33357886-33359585 cg14408969 C8orf40 NM_001135675; TSS1500; TSS1500; chr8: 42396235-42397195 NM_001135674; TSS1500; 5′UTR; NM_138436; NM_006749; TSS200 NM_001135676

Example 10. The Effect of Meditation on Genomic DNA Methylation, BDNF Level, and Cortisol Level

Table 2 illustrates the demographics of participants.

DEMOGRAPHICS Mean (SD) Range Gender 19 M:19 F Age (years) 34.28 (8.84) 21-59 Height (inches) 67.18 (4.20) 60-75 Weight (pounds) 142.26 (30.03)  96.2-216.0 Body Mass Index (BMI) (kg/m²) 22.05 (3.70) 17.04-34.38 Years of Yoga/Meditation Experience  4.54 (3.26) 0.2-15  Length of daily practice (minutes) 127.50 (41.22)  45-180

Table 3 illustrates the psychometrics including Brief Symptom Inventory (BSI) criteria, Freiburg Mindfulness, and Tellegen Absorption scale.

PSYCHOMETRICS Pre Mean Post Mean N = 34 (SD) (SD) t df p BSI-18 Total 79.5 (11.0) 86.9 (6.02) −4.66 33 <0.0001 BSI-Depression 26.9 (4.39) 28.7 (1.96) −2.84 33 <0.01 BSI-Anxiety 26.2 (4.20) 28.8 (2.07) −4.22 33 <0.0001 BSI-Somatic 26.3 (3.62) 28.4 (2.83) −4.66 33 <0.0001 Freiburg Mindfulness 39.6 (7.65) 44.5 (7.07) −4.42 33 <0.0001 Tellegen Absorption 88.6 (29.6) 91.3 (28.9) −0.86 33 0.4

Tables 4A and 4B illustrate the BDNF level from pre- and post-meditation sample.

TABLE 4A BIOMARKERS (n = 38) Pre Post Mean (SD) Mean (SD) Raw Ln Raw Ln t Valid N p B.M.I. 22.1 (3.7)  — 21.2 (3.1)  — 4.37 36 <0.0001 (kg/m²) BDNF 2513 (1484) 7.65 (0.64) 7039 (5274) 8.44 (1.12) −5.07 32 <0.0001 (pg/ml)

TABLE 4B BIOMARKERS (n = 38) Pre Post Mean (SD) Mean (SD) Raw Ln Raw Ln t Valid N p B.M.I. 24.1 (6.0) — 22.8 (4.9)  — 2.74 8 <0.05 (kg/m²) BDNF 2005 (747) 7.51 (0.48) 7629 (4649) 8.70 (0.82) 7.38 8 <0.0001 (pg/ml)

FIG. 35 shows the methylation age of 32 participants. Arrows going down (green): meditators with younger DNA at end of yoga intervention. Arrows going up (orange): meditators with older DNA at the end of yoga intervention. Blue line (dot) indicates meditators calendar age.

FIG. 36 shows the salivary cortisol level at 30 minutes after meditation either taken prior to attendance of a yoga retreat (Anaadhi yoga retreat) or post attendance of the yoga retreat.

Embodiment 1 comprises a method of increasing the expression rate of ELOVL2, KLF14, PENK, or a combination thereof in a first subject, comprising: (a) administering to the first subject a therapeutically effective dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof for a first time period; (b) obtaining a sample from the first subject; and (c) determining whether the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased in the first subject relative to a control by contacting the sample with a probe that recognizes ELOVL2, KLF14, or PENK and detecting binding between ELOVL2, KLF14, or PENK and the probe.

Embodiment 2 comprises the method of embodiment 1, wherein vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof is L-ascorbic acid 2-phosphate.

Embodiment 3 comprises the method of embodiment 1, wherein the expression level of ELOVL2 is determined by contacting the sample with a probe that recognizes ELOVL2 and detecting binding between the probe and ELOVL2.

Embodiment 4 comprises the method of embodiment 1, wherein the expression level of KLF14 is determined by contacting the sample with a probe that recognizes KLF14 and detecting binding between the probe and KLF14.

Embodiment 5 comprises the method of embodiment 1, wherein the expression levels of ELOVL2 and KLF14 are determined by contacting the sample with a probe that recognizes ELOVL2 and a probe that recognizes KLF14 and detecting each respective binding between the probes and ELOVL2 and KLF14.

Embodiment 6 comprises the method of embodiment 1, wherein the expression levels of ELOVL2, KLF14, and PENK are determined.

Embodiment 7 comprises the method of any one of the embodiments 1-6, wherein an increase in the expression rate of ELOVL2, KLF14, PENK, or a combination thereof further correlates to a decrease in cell senescence, an increase in cell proliferation, an increase in cell survival, or a decrease in DNA methylation.

Embodiment 8 comprises the method of any one of the embodiments 1-7, wherein an increase in the expression rate of ELOVL2, KLF14, PENK, or a combination thereof leads to a methylation pattern that mimics the methylation pattern of a sample obtained from a second subject.

Embodiment 9 comprises the method of embodiment 8, wherein the second subject is younger in chronological age relative to the first subject.

Embodiment 10 comprises the method of embodiment 8 or 9, wherein the second subject is younger in chronological age relative to the first subject by at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 50 years, or more.

Embodiment 11 comprises the method of embodiment 1, wherein the control comprises the expression level of ELOVL2, KLF14, PENK, or a combination thereof obtained from a sample from the subject prior to administration of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof.

Embodiment 12 comprises the method of embodiment 1, wherein the control comprises a normalized expression level of ELOVL2, KLF14, PENK, or a combination thereof obtained from a set of samples without exposure to vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof.

Embodiment 13 comprises the method of embodiment 12, wherein the set of samples are a set of cell samples.

Embodiment 14 comprises the method of embodiment 1, further comprising increasing the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has not increased relative to the control.

Embodiment 15 comprises the method of embodiment 1, further comprising increasing the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased relative to the control and at a rate that is below a target range.

Embodiment 16 comprises the method of embodiment 1, further comprising decreasing or maintaining the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof if the expression level of ELOVL2, KLF14, PEVK, or a combination thereof has increased relative to the control.

Embodiment 17 comprises the method of embodiment 1, further comprising maintaining the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased relative to the control and at a rate that is within a target range.

Embodiment 18 comprises the method of embodiment 1, further comprising decreasing the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof if the expression level of ELOVL2, KLF14, PENK, or a combination thereof has increased relative to the control and at a rate that is above a target range.

Embodiment 19 comprises the method of embodiment 14 or 16, wherein the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof is increased, decreased, or maintained for a second period of time prior to redetermining the expression level of ELOVL2, KLF14, PENK, or a combination thereof.

Embodiment 20 comprises the method of embodiment 1, wherein the first period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

Embodiment 21 comprises the method of embodiment 19, wherein the second period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

Embodiment 22 comprises the method of any one of the embodiments 1-21, further comprising determining the expression level of FHL2, SMC4, SLC125A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, BDNF, NDF, GDNF, cortisol, or a combination thereof.

Embodiment 23 comprises the method of any one of the embodiments 1-22, further comprising determining the expression level of an epigenetic marker selected from Table 1.

Embodiment 24 comprises a method of modulating the methylation pattern of ELOVL2, KLF14, PENK or a combination thereof in a first subject, comprising: (a) administering to the first subject a therapeutically effective dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof for a first time period; (b) obtaining a sample from the first subject; and (c) determining whether the methylation pattern of ELOVL2, KLF14, PENK or a combination thereof has changed in the first subject relative to a control by contacting the sample with a set of probes and detecting a set of hybridization products to determine the methylation pattern of ELOVL2, KLF14, PENK or a combination thereof.

Embodiment 25 comprises the method of embodiment 24, wherein vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof is L-ascorbic acid 2-phosphate.

Embodiment 26 comprises the method of embodiment 24, wherein the sample is further treated with a deaminating agent prior to determining the methylation pattern.

Embodiment 27 comprises the method of embodiment 24, wherein the methylation pattern of ELOVL2 is determined.

Embodiment 28 comprises the method of embodiment 24, wherein the methylation pattern of KLF14 is determined.

Embodiment 29 comprises the method of embodiment 24, wherein the methylation pattern of PENK is determined.

Embodiment 30 comprises the method of embodiment 24, wherein the methylation patterns of ELOVL2 and KLF14 are determined.

Embodiment 31 comprises the method of embodiment 24, wherein the methylation patterns of ELOVL2, KLF14, and PENK are determined.

Embodiment 32 comprises the method of any one of the embodiments 24-31, wherein a change in the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof is a decrease in methylation status of ELOVL2, KLF14, PENK, or a combination thereof.

Embodiment 33 comprises the method of embodiment 32, wherein a decrease in the methylation status of ELOVL2, KLF14, PENK, or a combination thereof further correlates to a decrease in cell senescence, an increase in cell proliferation, or an increase in cell survival.

Embodiment 34 comprises the method of embodiment 32, wherein a decrease in the methylation status of ELOVL2, KLF14, PENK, or a combination thereof leads to a methylation pattern that mimics the methylation pattern of a sample obtained from a second subject.

Embodiment 35 comprises the method of embodiment 34, wherein the second subject is younger in chronological age relative to the first subject.

Embodiment 36 comprises the method of embodiment 34 or 35, wherein the second subject is younger in chronological age relative to the first subject by at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 50 years, or more.

Embodiment 37 comprises the method of embodiment 24, wherein the control comprises the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof obtained from a sample from the subject prior to administration of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof.

Embodiment 38 comprises the method of embodiment 24, wherein the control comprises a normalized methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof obtained from a set of samples without exposure to vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof.

Embodiment 39 comprises the method of embodiment 38, wherein the set of samples are a set of cell samples.

Embodiment 40 comprises the method of embodiment 24, further comprising increasing the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has not changed relative to the control.

Embodiment 41 comprises the method of embodiment 24, further comprising increasing the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control and to a degree lower than a target range.

Embodiment 42 comprises the method of embodiment 24, further comprising decreasing or maintaining the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control.

Embodiment 43 comprises the method of embodiment 24, further comprising maintaining the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control and to a degree within a target range.

Embodiment 44 comprises the method of embodiment 24, further comprising decreasing the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof if the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof has changed relative to the control and to a degree above a target range.

Embodiment 45 comprises the method of embodiment 40 or 41, wherein the dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof is increased, decreased, or maintained for a second period of time prior to redetermining the methylation pattern of ELOVL2, KLF14, PENK, or a combination thereof.

Embodiment 46 comprises the method of embodiment 24, wherein the first period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

Embodiment 47 comprises the method of embodiment 43, wherein the second period of time comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.

Embodiment 48 comprises the method of any one of the embodiments 24-47, further comprising determining the methylation pattern of FHL2, SMC4, SLC12A5, TEZM151A, TTF2, TRIM45, TRIM59, ACSS3, ARID5A, BLMH, BRD4, CD28, EPHX3, RIN1, SLX1, BDNF, NDF, GDNF, cortisol, or a combination thereof.

Embodiment 49 comprises the method of any one of the embodiments 24-48, further comprising determining the methylation pattern of an epigenetic marker selected from Table 1.

Embodiment 50 comprises the method of any one of the embodiments 1-49, wherein the therapeutically effective dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof comprises from about 0.1 μg/mL to about 200 μg/mL, from about 1 μg/mL to about 150 μg/mL, from about 5 μg/mL to about 100 μg/mL, from about 10 μg/mL to about 100 μg/mL, from about 20 μg/mL to about 100 μg/mL, from about 30 μg/mL to about 100 μg/mL, from about 50 μg/mL to about 100 μg/mL, from about 1 μg/mL to about 50 μg/mL, from about 5 μg/mL to about 50 μg/mL, from about 10 μg/mL to about 50 μg/mL, or from about 50 μg/mL to about 200 μg/mL.

Embodiment 51 comprises the method of any one of the embodiments 1-49, wherein a dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof greater than 200 μg/mL increases reactive oxidative species.

Embodiment 52 comprises the method of any one of the embodiments 1-49, wherein a dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof greater than 200 μg/mL leads to a methylation pattern that mimics the methylation pattern of a sample obtained from a third subject who is older in chronological age relative to the first subject.

Embodiment 53 comprises the method of embodiment 52, wherein the third subject is older in chronological age relative to the first subject by at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 50 years, or more.

Embodiment 54 comprises the method of any one of the embodiments 1-53, wherein the probe hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1-199.

Embodiment 55 comprises the method of any one of the embodiments 1-56, further comprising administering to the first subject an additional therapeutic agent.

Embodiment 56 comprises the method of any one of the embodiments 1-55, wherein the sample is obtained from a subject having a metabolic disease or condition.

Embodiment 57 comprises the method of embodiment 56, wherein the metabolic disease or condition comprises diabetes or pre-diabetes.

Embodiment 58 comprises the method of embodiment 57, wherein diabetes is type I diabetes, type II diabetes, or type IV diabetes.

Embodiment 59 comprises the method of any one of the embodiments 1-55, wherein the sample is obtained from a subject having a ELOVL2-associated disease or indication, a KLF14-associated disease or indication, or a PENK-associated disease or indication.

Embodiment 60 comprises the method of any one of the embodiments 1-55, wherein the sample is obtained from a subject having Werner syndrome, progeria, or post-traumatic stress disorder.

Embodiment 61 comprises the method of any one of the embodiments 1-55, wherein the sample is obtained from a subject having an elevated body mass index (BMI).

Embodiment 62 comprises the method of embodiment 61, wherein the elevated BMI is a BMI of 25 kg/m², 26 kg/m², 27 kg/m², 28 kg/m², 29 kg/m², 30 kg/m², 35 kg/m², 40 kg/m² or more.

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. 

What is claimed is:
 1. A method for treating a subject with vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof, the method comprising the steps of: administering to the subject an initial dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof for a first time period; determining whether the subject's expression of ELOVL2 has increased after the administration of the initial dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof by: obtaining a sample from the subject; and performing an analysis of the sample to determine if the subject's current level of expression of ELOVL2 after the administration of the initial dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof is greater than the subject's previous level of expression of ELOVL2 before the administration; and if the subject's expression of ELOVL2 has increased, administering to the subject a revised dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof that is equal to or lower than the initial dose; or if the subject's expression of ELOVL2, has not increased, administering to the subject a revised dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof, that is greater than the initial dose, to no more than 200 μg/mL.
 2. The method of claim 1, wherein the analysis comprises contacting the sample with a nucleic acid probe which hybridizes to ELOVL2 and detecting binding between ELOVL2 and the nucleic acid probes.
 3. The method of claim 2, wherein the nucleic acid probes hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1, 5, and
 21. 4. The method of claim 1, wherein the first time period comprises at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, 1 year, two years, three years, or more.
 5. The method of claim 1, wherein vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof is L-ascorbic acid 2-phosphate.
 6. The method of claim 1, wherein the method further comprises detecting a level of cortisol in the subject after the administration of the initial dose of vitamin C or its derivatives, metabolites, or pharmaceutically acceptable salts thereof.
 7. The method of claim 1, wherein the sample is obtained from a subject having a metabolic disease or condition.
 8. The method of claim 1, wherein the sample is obtained from a subject having diabetes.
 9. The method of claim 1, wherein the sample is obtained from a subject having Werner syndrome, progeria, or post-traumatic stress disorder.
 10. The method of claim 1, wherein the sample is a cell sample. 